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Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode
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Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode Madhavi Bandapati a, Prabhat K. Dwivedi b,***, Balaji Krishnamurthy a,**, Young Ho Kim c, Gyu Man Kim d, Sanket Goel e,* a
Department of Chemical Engineering, Birla Institute of Technology and Science (BITS) Pilani, Hyderabad Campus, Hyderabad, India b Center for Nanosciences, Indian Institute of Technology (IIT) Kanpur, Kanpur, India c Medical Device Development Centre, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, South Korea d School of Mechanical Engineering, Kyungpook National University, Daegu 41566, South Korea e Department of Electrical and Electronics Engineering, Birla Institute of Technology and Science (BITS) Pilani, Hyderabad Campus, Hyderabad, India
article info
abstract
Article history:
Research in Enzymatic Biofuel cells (EBFCs) has evolved during the last decade. However,
Received 10 May 2017
challenges, such as cost and endurance are the bottlenecks in harnessing their commer-
Received in revised form
cialization potential. To overcome these challenges, low cost, readily available pencil leads
14 July 2017
of various grades modified with polyaniline (PANI) and multi walled carbon nano tubes
Accepted 4 September 2017
(MWCNT) have been examined as EBFC biocathode. Total four pencils of various grades (B,
Available online xxx
H, 3H, and 5H) were used to fabricate biocathode by covalently immobilizing Laccase(Lac) enzyme on to MWCNT coated (BC1) and electrodeposited polyaniline and MWCNT coated
Keywords:
(BC2) on the surface of pencil graphite electrodes (PGEs). The fabricated biocathodes were
Enzymatic biofuel cell (EBFC)
characterized by scanning electron microscopy (SEM), BET surface area and conductivity
Biocathode
measurements and electrochemical analysis was performed by the Open circuit potential
Electro-polymerization
(OCP) and Cyclic voltammetry (CV). Among the pencils tested in this work, the 5H pencil
Polyaniline (PANI)
coated with PANI/MWCNT/Lacexhibited highest current density of 1209.23 mA/cm2 with
Multi-walled carbon nanotube
OCP of 0.528 V. Moreover, the PANI/MWCNT/Lac modified PGEs showed significant
(MWCNT)
enhancement (80%) in electrochemical behavior when compared with unmodified (bare)
Mediated electron transfer (MET)
and MWCNT/Lac coated PGE. Both types of Bioelectrodes (PGE/MWCNT/Lac,PGE/PANI/ MWCNT/Lac) showed good stability and maximum conservation of its activity during the experiments. © 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (P.K. Dwivedi), [email protected] (B. Krishnamurthy), sgoel@hyderabad. bits-pilani.ac.in (S. Goel). https://doi.org/10.1016/j.ijhydene.2017.09.016 0360-3199/© 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016
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Introduction Enzymatic Biofuel Cell (EBFC), a bioelectronic device, is a subset of biofuel cell that utilizes redox enzymes as the electro-catalyst to generate electric current from organic molecules [1,2]. It consists of bioanode, where the fuel such as sugars and alcohols are electro oxidized at reducing potential with the help of anodic enzymes like Glucose Oxidase and Glucose Dehydrogenase and the biocathode where dioxygen is electro reduced at an oxidizing potential with cathodic enzyme like Laccase, Bilirubin Oxidase and Tyrosinase [3e5]. The specific and selective nature of catalytic reactions triggered by the use of enzymes as biocatalyst eliminates the need for a membrane separator leading to the possibility to use a single chamber for anodic and cathodic half-cell reactions [6,7]. Further it promotes the cell to function under normal working conditions (ambient temperature, neutral pH) with a wide range of organic materials as fuels and zero emission of greenhouse gases [8]. Due to the high availability of these fuels in biological and environmental systems, the use of EBFCs as uninterrupted power source is being explored for portable electronic devices in many fields including medicine and military [9e13]. However, lower current density, short life span of the enzymes and high operating and maintenance cost are the key obstacles for the commercialization of EBFCs [14]. To address these issues, in the recent years, extensive research has been performed to understand the enzyme catalytic reaction mechanism [15,16], on electrode modification [17], developing new bio-material [18e21], various ways for enzyme immobilization and enzyme electrode assemblies [22,23]. It is found that selection of proper electrode materials and enhancing enzyme immobilization to achieve high enzyme loading improves the performance of EBFCs. Electrical conductivity and hardness are the two main factors to consider while selecting the best electrode material [24,25]. Many novel electrode materials and modifications have been proposed, particularly harnessing off-the-shelf available pencil graphite electrodes has been demonstrated to be excellent material for biocathode [26,27]. Further, pencil graphite electrodes (PGEs) bring other added advantages, such as good mechanical rigidity, high electrochemical reactivity, easy disposal, low cost, flexibility of modifications [28,29]. These useful properties of inexpensive pencil graphite electrodes have led to their use in analytical applications by many scientists in the recent years. They have been used for determination of hemoglobin [30], measuring vitamin C content of commercial orange juice [31], measurement of DNA and RNA [32e35],determination of catecholamines in blood [36,37], determination of a polycyclic aromatic carcinogen [38], detection of cyclophosphamide using DNA [39], immobilization and detection characterization of micro particle of a human gene [40,41], determination of trace metals [42e44], analysis of salicylic acid in plant Material [45], as a solid-phase micro extraction fiber [46,47], monitoring caffeine levels in tea samples using the squarewave anodic stripping voltammetry (SWASV) method [48], production of hydrogen from water using modified pencil graphite electrodes [49]. In our previous study [50], pencil graphite was used for fabrication of biocathode by modifying
surface with PANI and MWCNT for enzymatic biofuel cell application. The results showed that biocathode prepared with PGE exhibited enhanced stability towards oxygen reduction reaction. The present work aims to select the more efficient pencil among commercially available grades of pencil to build an efficient biocathode for EBFC application with mediated electron transfer (MET) between the active sites of the electrode surface and enzymes. This was performed by first studying and screening the surface characteristics and electrochemical properties of pencil graphite of various grades modified with MWCNT and PANI coatings. Thereafter, the results were compared with unmodified (bare) pencil graphite, to determine the best pencil graphite as EBFC biocathode. The work focuses on the realization of a biocathode by substituting the traditional, precious and costly Platinum metal catalyst with an enzymatic catalyst system.
Experimental Materials All reagents were procured from Sigma Aldrich. Laccase from Trametes Versicolor (E.C. 1.10.3.2, 24 U/mg) was stored at 20 C. Other chemicals were Monosodium phosphate Disodium phosphate (Na2HPO4), Aniline (NaH2PO4), (C6H5NH2), 2, 2- azino-bis (3-ethylbenzothiazoline-6sulphonic acid) (ABTS), Dimethyl sulfoxide (DMSO), 1-Ethyl3-(3-dimethylaminopropyl) carbodiimide (EDC), Multi-Walled Carbon nanotube carboxylic acid functionalized(MWCNTCOOH, average diameter Length, 9.5 nm 1.5 mm), N-hydroxy succamide (NHS), Hydrochloric acid (HCl) and Acetone. The pencil leads tested were B, H, 3H, and 5H, purchased from a local bookstore, of 60 mm length and varying diameters (2 mm, 1.82 mm, 1.9 mm and 1.88 mm for pencil leads B, H, 3H and 5H respectively). The supporting electrolyte phosphate buffer solution (PBS 0.1 M, pH 5.0) was arranged from NaH2PO4 and Na2HPO4 for biocathode electrochemical analysis. For all the experiments, ultrapure water with resistivity of 18.2 M U cm was used.
Apparatus BET surface area analyzer Smartsorb 93 type surface area analyzer was used to calculate surface area of modified PGEs by dynamic BrunauereEmmetteTeller(BET) principle. The Nitrogen gas used for adsorption and different gas mixture percentages were used to measure total pore volume and surface area. Prior to the measurements, all of the samples were degassed at 473 K for 2 h. The user friendly software stored the data and computed the final surface area and pore volume values from adsorption desorption curves displayed on the screen.
Potentiostat/galvanostat The electrodeposition of PANI and electrochemical analysis were accomplished using a computer controlled Auto lab PGSTAT-302Ntype Potentiostat (Metrohm Auto lab, the Netherlands) driven by NOVA 1.11 software (Eco Chemie, the
Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016
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Netherlands). The experiments were carried out in a conventional three electrode cell (25 ml) under an aerobic condition, composed of working electrode (modified PGEs), Ag/AgCl (3 M KCl) reference electrode and a Platinum wire counter electrode.
Scanning electrode microscope (SEM) The morphological studies of modified PGEs were examined using ZEISS Supra 40VP SUPRA 40 VP Field Emission Scanning Electron Microscope.
Conductivity measurements The conductivity of modified and unmodified pencil leads was calculated using digital multivoltmeter (Mastech MAS830L). The PGEs (bare or modified) were placed in a conducting device which consists of the brass stamp electrodes and the distance between them is maintained consistent by tightening the compression screws. A digital multimeter was used to measure the resistance across the two brass stamp electrodes. The standard equation was used to find the resistivity from resistance values of the modified and unmodified PGEs. Resistivity (r) ¼ R.A/d where A is the effective area of the measuring electrode and d is the distance between two measuring electrodes. Inverse of resistivity is the conductivity.
Procedure Pretreatment of pencil graphite electrodes (PGE) The wooden part of pencils was carefully peeled out with a razor blade. The obtained pencil lead rods were cleaned with 0.5 M HCl solution in ultra-sonication bath for 2 min. Subsequently, they were washed and sonicated in sequence with the deionized water for few times and in acetone for a couple of minutes respectively, and kept for drying in a lab oven for 2 h at 90 C.
Electropolymerization of aniline Pretreated PGEs were used to perform electropolymerization of aniline using cyclic voltammetry (CV) in the range 0.2 V to 1 V at 50 mV/s scanrate. To prepare the electrolyte solution for aniline electropolymerization, 10 ml of aniline was dissolved in the solution of 100 ml of 1.0 M HCl. To get rid of surplus unbound PANI, after polymerization, the PGEs were washed with deionized water. The electrodeposition of PANI on PGE involved the standard steps - adsorption, nucleation, and polymerization.
Preparation of PGE/MWCNT (BC1) electrode Deposition of MWCNT on PGE surface was done by dip coating. The MWCNT-COOH (originally purchased as 1 mg/ml) was isolated in DMSO solvent by ultra-sonication for 2 min. Each pretreated bare PGE was immersed in prepared MWCNTCOOH dispersion for complete wetting and to get sufficient interaction of PGEs with MWCNT solution. A film of MWCNTCOOH deposits on surface of PGEs. Thereafter, the samples were kept in hot air oven (2 h at 90 C) to evaporate solvent
3
from their surface. This procedure was repeated 4 times to deposit adequate amount of MWCNT on PGE surface.
Preparation of PGE/PANI/MWCNT (BC2) electrode First, 30 mM EDC and 90 mM NHS were dissolved in PBS, and then MWCNT solution was prepared (MWCNT in EDC/NHS in 1:1 ratio) and the PANI coated PGEs were immersed into MWCNT solution for 2 h. Thereafter, by bioconjugate technique, the carboxylated MWCNT were covalently attached to the free amine group of PANI by amide bond. EDC/NHS compounds were used to activate carboxylic group in organic solvents.
Fabrication of biocathode from modified PGEs First, Laccase enzyme solution was prepared by mixing 3 mg/ml Laccase in 0.1 M PBS. Biocathode were developed by dispersing PGE/MWCNT (BC1) and PGE/PANI/MWCNT(BC2) modified electrodes into the Laccase enzyme solution and letting it remain at room temperature for 24 h. The enzyme was covalently attached to the activated carboxylic group of MWCNT by amide bond on modified electrodes. Later, fabricated biocathodes were washed with PBS to washout unbound enzymes.
Electrochemical measurements Electrochemical characterization of unmodified (bare) and modified PGEs was performed by open circuit potential (OCP) and cyclic voltammetry (CV) techniques. The supporting electrolyte to conduct these tests was PBS (0.1 M, pH 5.0) with 1 mM which ABTS as cathodic mediator [52]. MET is preferred over DET in this study as only a few enzymes are capable of DET [10,53]. All measurements were carried out with a scan rate of 50 mV/s at the room temperature in air saturated buffer solution.
Storage of Laccase immobilized biocathode The fabricated biocathode immobilized with Laccase enzyme on modified PGEs were stored in PBS at 4 C.
Results and discussions Surface characteristics of modified PGEs Scanning electron microscopy (SEM) The surface morphology of pencil graphite electrodes fabricated from 5H pencil leads were characterized by Scanning Electron Microscopy at each step of the fabrication. Fig. 1 shows the SEM image of bare PGE at 5 and 100 magnifications, clearly indicating smooth, layered and featureless morphology with some irregular surface depressions. Fig. 2a shows the SEM image of PGE modified with MWCNT by dip coating. It shows non-uniform distribution of nanotubes developed by their intermolecular interaction and mechanical linking. They appear to be arranged in the form of wires or packets with close packed stacking through van der waals force. Fig. 2b shows SEM image of the surface of PGE modified
Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016
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Fig. 1 e SEM images of pretreated unmodified PGEs from 5H pencil at varying resolutions (a) 5£ (b) 100£.
Fig. 2 e SEM images of (a) PGE/MWCNT (BC1) (b) PGE/PANI/MWCNT (BC2) electrodes for 5H pencil.
with PANI/MWCNT. As can be seen, even though the surface morphology was similar, the MWCNT attached to the nanoporous background PANI by covalent bond shows the presence of thick dense matrix of nanotubes. This offers more free carboxylic groups for high loading of enzymes on to the
surface by amide bond. In Fig. 3a, the SEM images for Laccase immobilized on the PGE coated with MWCNT show the formation of enzyme agglomerates. Although the dispersion of nanotubes on PGE increases the surface area, the random and non-uniform dispersion of nanotubes on the surface of 5H PGE
Fig. 3 e SEM images of (a) PGE/MWCNT (BC1), (b) PGE/PANI/MWCNT (BC2) electrode with immobilized Laccase enzyme for 5H pencil. Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016
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resulted in the ineffective dispersion of the enzymes. Fig. 3b, where Laccase was immobilized on PG/PANI/MWCNT, shows uniform dispersion of the enzymes. Thus, the modification of the PGE with the conducting materials of PANI and MWCNT resulted in greater immobilization of Laccase on the electrode and increased the availability of the enzymes for catalytic activity. Although the PGE was optimized by coating it with conducting material, the chemical and structural properties of various pencil leads also contribute to the electrode properties. The diverse morphological characteristics of various leads were due to their varying composition in terms of graphite content, polymers and clay content [Table 1]. PGEs fabricated from various grades of pencil leads were therefore studied and the electrode properties were found to be optimal for 5H PGE for which the SEM results have been discussed in detail.
Conductivity measurement Electrical conductivity of modified and unmodified(bare) PGEs was measured at room temperature and results are summarized in Table 1. In case of bare PGEs, the conductivity is found to be increasing with increasing graphite percentage of Pencil leads. The highest conductivity recorded for B pencil can be attributed to the highest content of graphite (71%) (See Table 1). The PGEs coated with MWCNT showed increased electrical conductivity upon bare PGEs due to the conducting nature of MWCNT [54]. However, PGEs upon coating with PANI and MWCNT resulted in lower conductivity than that of bare PGE and MWCNT coated PGEs. The reduction in conductivity is due to the random and mixed graphite-insulating structure and with the random assembly of graphite particles inside the rigid non-conducting matrix of binding material (clay and wax).
BET surface area and pore volume measurement The specific surface area (SBET) and pore volume (SPV) of pencil graphite electrodes fabricated from various grades of pencil leads were calculated at each step of the fabrication. The results are shown in Table 1. The bare 5H PGE showed higher surface area with high pore volume distribution. 5H pencil is the hardest pencil among the tested pencils with high content of clay (42%) causing more disorder with high pore volume distribution and surface area, since clay contributes to increase the disorder in the host layered structure of graphite [29]. Further, very less surface area and Pore volume distribution was observed for PANI coated 5H PGE confirming the
formation of thick film of conducting polymer on the surface. The pore volume and surface area for tested pencils (H, 3H, 5H, and B) are shown in Fig. 4.
Electrochemical characterization of PGE The electrochemical performance of unmodified (bare) and modified (PGE/MWCNT/Lac, PGE/PANI/MWCNT/Lac) electrodes were examined. In order to characterize the fabricated biocathode, Open circuit potential (OCP) and Cyclic voltammetry (CV) based analysis were carried out for oxygen reduction reaction.
Open circuit potential (OCP) The OCP of fabricated biocathodes from various grades of pencil leads were measured in air saturated PBS with Ag/AgCl as reference electrode, Platinum wire as counter electrode. Table 2 summarizes the OCP values calculated for 300 s. The measured values were very close to the reduction potential of dioxygen. From the results, it was observed that among the tested pencils, B pencil showed highest OCP for both bare and modified electrodes owing to the higher graphite content (Table 1). Slight reduction in OCP was observed for the same pencil when coated with MWCNT and PANI/MWCNT. This may be due to the blocking of MWCNT with non-conducting binder material used for manufacturing of pencils and also the varying structural properties of PGE surfaces. Further, lower OCP was observed for bare 5H pencil without any modification due to its less graphite content (Table 1). Subsequently, the pencil leads were modified with conducting material MWCNT and PANI. With BET surface area measurements (Table 1), it was observed that 5H pencil possessed highest surface area and pore volume distribution, which facilitated more amount of MWCNT on the surface of 5H pencil. Such modification led to high electron transfer rate, attributed to large OCP compared to unmodified PGE. Comparative graphs for all pencils are shown in Fig. 5 for unmodified (bare) and modified PGEs for all the four pencils.
Cyclic voltammetry (CV) Cyclic Voltammetry of bare and modified (PGE/MWCNT/Lac (BC1), PGE/PANI/MWCNT/Lac (BC2))electrodes was recorded in the voltage range 1.0 V to 1.0 V at 50 mV/s scan rate [52] and substantial difference was observed in their response The obtained current densities calculated from geometrical surface area of pencil leads are tabulated in Table 2. All the
Table 1 e Surface characteristics of unmodified (bare) and modified PGEs. S. no
1 2 3 4 *
Pencil type
Graphite (%)
Clay (%)
H 3H 5H B
63 58 52 71
31% 36% 42% 23%
SBET(m2/gr)
SPV (cm3/gr)
Conductivity (S/cm)
bare
BC1
BC2
bare
BC1
BC2
bare
BC1
BC2
2.23 3.67 5.63 3.15
2.13 2.86 6.26 3.87
0.01 0.08 1.79 3.02
0.0034 0.0024 0.0093 0.0046
0.0017 0.0032 0.0074 0.0056
0.0001 0.0002 0.0045 0.0042
876 922 390 121247
1071 1090 405 142784
1045 653 401 445
BC1 e PGE coated with MWCNT *BC2 —PGE coated with PANI/MWCNT.
Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016
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Fig. 4 e Comparative graphs of (a) pore volume (SPV) and (b) surface area (SBET) of all tested pencils.
modified PGEs showed well defined voltammetric peaks at a peak potential close to the redox potential of T1 copper active site determined for Trametes Versicolor Laccase where the ABTS mediator oxidized and transferred electrons to trinuclear copper center T2/T3 of Laccase enzymes. The fully reduced Laccase enzyme reacted with dioxygen and converted into water [51,55]. Thus confirming the catalytic character of Laccase enzyme towards the O2/H2O redox couple (see Fig. 6). From CV studies, the low peak currents observed for bare PGEs are indicative of minimal reduction reaction occurring in the absence of Laccase catalyst. Further, 5H PGE which has the least graphite content among tested pencil leads has
registered least response towards electrochemical reaction. The CV of BC2 (Fig. 8) electrode exhibited enhanced current response than BC1 (Fig. 7) and bare PGE. As mentioned in Table 1, for 5H BC2 (with PANI/MWCNT), the surface area and pore volume distribution reduced (in comparison to bare pencil and BC1). This confirmed the electrochemical deposition of thick layer of highly conducting Emeraldine salt form of polyaniline (PANI) film on electrode surfaces [56]. Such scheme has dual advantage e first, it supports for high peak currents due to the enhanced overall conductivity, and second, the composite layer ensures direct electron transfer (DET) process to the modified electrode surfaces along with ABTS cathodic mediator.
Table 2 e Electrochemical Characteristics of unmodified (bare) and modified PGEs. S. No
1 2 3 4
Pencil type
H 3H 5H B
Current density (mA/cm2)
OCP (vs. Ag/AgCl) (V) Bare
BC1
BC2
Bare
BC1
BC2
0.477 0.516 0.467 0.578
0.571 0.536 0.574 0.517
0.583 0.573 0.528 0.564
51.724 66.260. 8.372 33.616
15.138 11.862 62.790 70.771
436.140 231.912 1209.236 503.263
Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016
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Fig. 5 e Comparative studies of obtained OCP values for four (H, 3H, 5H and B) pencils unmodified (bare) and modified.
From comparative studies, 5H pencil modified with PANI/MWCNT showed highest current density 1209 mA/ cm2. The highest current response of 5H pencil attribute to the high carboxylic groups on surface with MWCNT as confirmed from SEM images. Table 2 shows the peak currents for all the PGEs, studied in this work, both
unmodified and modified. Observation of overall trend on obtained results for various PGEs, the varying amount of graphite content in pencil leads and composition of the binding material like clay and pencil hardness are the main cause of the difference in response characteristics of PGEs.
600 500
5H
3H
H
B
400 300
Current(μA)
200 100 0 -100 -200 -300 -400 -500 -0.2
0
0.2
0.4 0.6 Applied Potential (V)
0.8
1
1.2
Fig. 6 e Cyclic voltammetry of unmodified (bare) PGEs (H, 3H, 5H and B).
Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016
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2000 B
5H
3H
H
1500
Current(μA)
1000
500
0
-500
-1000
-1500 -0.4
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
Applied Voltage(V) Fig. 7 e Cyclic voltammetry of PGE/MWCNT (BC1) electrode immobilized with Laccase enzyme for four pencils (H, 3H, 5H and B).
Fig. 8 e Cyclic voltammetry of PGE/PANI/MWCNT (BC2) electrode immobilized with Laccase enzyme for four pencils (H, 3H, 5H and B). Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016
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Conclusions Cost effective biocathodes, PGE/MWCNT (BC1) and PGE/PANI/ MWCNT (BC2) immobilized with Laccase enzymes, were fabricated by screening various readily available grades of pencil leads with different carbon content. Morphological characterization confirmed micro-and-nanoporous structures proving uniform distribution of the immobilized Laccase enzyme. While various tested pencils exhibited different response characteristics towards oxygen reduction reaction, any trend among the pencils with respect to graphite was not experiential. However, the electrochemical behavior of the pencil graphite electrode (PGEs) was found to be greatly influenced by the different compositions, such as clay and other binder components, leading to their disordered structure and pencil hardness. CV and OCP studies indicate that 5H pencil coated with PANI and MWCNT have highest potential for use as biocathode in EBFC.
Acknowledgement The work was financially supported from the India-Korea Project by the Department of Science and Technology, Government of India (INT/Korea/P-18/2013) to SG, the Research Initiation Grant from BITS-Pilani, Hyderabad Campus to SG, and the Nanomission project by the Department of Science and Technology, Government of India (SR/NM/NS-82/2016 (G)) to PKD.
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[33] Erdem A, Papakonstantinou P, Murphy H. Direct DNA hybridization at disposable graphite electrodes modified with carbon nanotubes. Anal Chem 2006;78(18):6656e9. [34] Wang J, Kawde AN. Pencil-based renewable biosensor for label-free electrochemical detection of DNA hybridization. Anal Chim Acta 2001;431(2):219e24. € € o € z M, Turan S. Electrochemical [35] Ozcan A, S‚ahin Y, Ozs oxidation of ds-DNA on polypyrrole nanofiber modified pencil graphite electrode. Electroanalysis 2007;19(21):2208e16. [36] Perati PR, Cheng J, Jandik P, Hanko VP. Disposable carbon electrodes for liquid chromatographic detection of catecholamines in blood plasma samples. Electroanalysis 2010;22(3):325e32. € [37] Ozcan A, S‚ahin Y. Selective and sensitive voltammetric determination of dopamine in blood by electrochemically treated pencil graphite electrodes. Electroanalysis 2009;21(21):2363e70. € o € z M, S‚entu¨rk Z. [38] Yardım Y, Keskin E, Levent A, Ozs Voltammetric studies on the potent carcinogen, 7, 12dimethylbenz [a] anthracene: adsorptive stripping voltammetric determination in bulk aqueous forms and human urine samples and detection of DNA interaction on pencil graphite electrode. Talanta 2010;80(3):1347e55. [39] Palaska P, Aritzoglou E, Girousi S. Sensitive detection of cyclophosphamide using DNA-modified carbon paste, pencil graphite and hanging mercury drop electrodes. Talanta 2007;72(3):1199e206. [40] Perdicakis M, Aubriet H, Walcarius A. Use of a commercially available wood-free resin pencil as convenient electrode for the ‘voltammetry of microparticles’ technique. Electroanalysis 2004;16(24):2042e50. [41] Hejazi MS, Alipour E, Pournaghi-Azar MH. Immobilization and voltammetric detection of human interleukine-2 gene on the pencil graphite electrode. Talanta 2007;71(4):1734e40. [42] Demetriades D, Economou A, Voulgaropoulos A. A study of pencil-lead bismuth-film electrodes for the determination of trace metals by anodic stripping voltammetry. Anal Chim Acta 2004;519(2):167e72. [43] Bond AM, Mahon PJ, Schiewe J, Vicente-Beckett V. An inexpensive and renewable pencil electrode for use in fieldbased stripping voltammetry. Anal Chim Acta 1997;345(1e3):67e74. [44] Majidi MR, Asadpour-Zeynali K, Hafezi B. Reaction and nucleation mechanisms of copper electrodeposition on
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Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode Madhavi Bandapati a, Prabhat K. Dwivedi b,***, Balaji Krishnamurthy a,**, Young Ho Kim c, Gyu Man Kim d, Sanket Goel e,* a
Department of Chemical Engineering, Birla Institute of Technology and Science (BITS) Pilani, Hyderabad Campus, Hyderabad, India b Center for Nanosciences, Indian Institute of Technology (IIT) Kanpur, Kanpur, India c Medical Device Development Centre, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, South Korea d School of Mechanical Engineering, Kyungpook National University, Daegu 41566, South Korea e Department of Electrical and Electronics Engineering, Birla Institute of Technology and Science (BITS) Pilani, Hyderabad Campus, Hyderabad, India
article info
abstract
Article history:
Research in Enzymatic Biofuel cells (EBFCs) has evolved during the last decade. However,
Received 10 May 2017
challenges, such as cost and endurance are the bottlenecks in harnessing their commer-
Received in revised form
cialization potential. To overcome these challenges, low cost, readily available pencil leads
14 July 2017
of various grades modified with polyaniline (PANI) and multi walled carbon nano tubes
Accepted 4 September 2017
(MWCNT) have been examined as EBFC biocathode. Total four pencils of various grades (B,
Available online xxx
H, 3H, and 5H) were used to fabricate biocathode by covalently immobilizing Laccase(Lac) enzyme on to MWCNT coated (BC1) and electrodeposited polyaniline and MWCNT coated
Keywords:
(BC2) on the surface of pencil graphite electrodes (PGEs). The fabricated biocathodes were
Enzymatic biofuel cell (EBFC)
characterized by scanning electron microscopy (SEM), BET surface area and conductivity
Biocathode
measurements and electrochemical analysis was performed by the Open circuit potential
Electro-polymerization
(OCP) and Cyclic voltammetry (CV). Among the pencils tested in this work, the 5H pencil
Polyaniline (PANI)
coated with PANI/MWCNT/Lacexhibited highest current density of 1209.23 mA/cm2 with
Multi-walled carbon nanotube
OCP of 0.528 V. Moreover, the PANI/MWCNT/Lac modified PGEs showed significant
(MWCNT)
enhancement (80%) in electrochemical behavior when compared with unmodified (bare)
Mediated electron transfer (MET)
and MWCNT/Lac coated PGE. Both types of Bioelectrodes (PGE/MWCNT/Lac,PGE/PANI/ MWCNT/Lac) showed good stability and maximum conservation of its activity during the experiments. © 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (P.K. Dwivedi), [email protected] (B. Krishnamurthy), sgoel@hyderabad. bits-pilani.ac.in (S. Goel). https://doi.org/10.1016/j.ijhydene.2017.09.016 0360-3199/© 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016
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Introduction Enzymatic Biofuel Cell (EBFC), a bioelectronic device, is a subset of biofuel cell that utilizes redox enzymes as the electro-catalyst to generate electric current from organic molecules [1,2]. It consists of bioanode, where the fuel such as sugars and alcohols are electro oxidized at reducing potential with the help of anodic enzymes like Glucose Oxidase and Glucose Dehydrogenase and the biocathode where dioxygen is electro reduced at an oxidizing potential with cathodic enzyme like Laccase, Bilirubin Oxidase and Tyrosinase [3e5]. The specific and selective nature of catalytic reactions triggered by the use of enzymes as biocatalyst eliminates the need for a membrane separator leading to the possibility to use a single chamber for anodic and cathodic half-cell reactions [6,7]. Further it promotes the cell to function under normal working conditions (ambient temperature, neutral pH) with a wide range of organic materials as fuels and zero emission of greenhouse gases [8]. Due to the high availability of these fuels in biological and environmental systems, the use of EBFCs as uninterrupted power source is being explored for portable electronic devices in many fields including medicine and military [9e13]. However, lower current density, short life span of the enzymes and high operating and maintenance cost are the key obstacles for the commercialization of EBFCs [14]. To address these issues, in the recent years, extensive research has been performed to understand the enzyme catalytic reaction mechanism [15,16], on electrode modification [17], developing new bio-material [18e21], various ways for enzyme immobilization and enzyme electrode assemblies [22,23]. It is found that selection of proper electrode materials and enhancing enzyme immobilization to achieve high enzyme loading improves the performance of EBFCs. Electrical conductivity and hardness are the two main factors to consider while selecting the best electrode material [24,25]. Many novel electrode materials and modifications have been proposed, particularly harnessing off-the-shelf available pencil graphite electrodes has been demonstrated to be excellent material for biocathode [26,27]. Further, pencil graphite electrodes (PGEs) bring other added advantages, such as good mechanical rigidity, high electrochemical reactivity, easy disposal, low cost, flexibility of modifications [28,29]. These useful properties of inexpensive pencil graphite electrodes have led to their use in analytical applications by many scientists in the recent years. They have been used for determination of hemoglobin [30], measuring vitamin C content of commercial orange juice [31], measurement of DNA and RNA [32e35],determination of catecholamines in blood [36,37], determination of a polycyclic aromatic carcinogen [38], detection of cyclophosphamide using DNA [39], immobilization and detection characterization of micro particle of a human gene [40,41], determination of trace metals [42e44], analysis of salicylic acid in plant Material [45], as a solid-phase micro extraction fiber [46,47], monitoring caffeine levels in tea samples using the squarewave anodic stripping voltammetry (SWASV) method [48], production of hydrogen from water using modified pencil graphite electrodes [49]. In our previous study [50], pencil graphite was used for fabrication of biocathode by modifying
surface with PANI and MWCNT for enzymatic biofuel cell application. The results showed that biocathode prepared with PGE exhibited enhanced stability towards oxygen reduction reaction. The present work aims to select the more efficient pencil among commercially available grades of pencil to build an efficient biocathode for EBFC application with mediated electron transfer (MET) between the active sites of the electrode surface and enzymes. This was performed by first studying and screening the surface characteristics and electrochemical properties of pencil graphite of various grades modified with MWCNT and PANI coatings. Thereafter, the results were compared with unmodified (bare) pencil graphite, to determine the best pencil graphite as EBFC biocathode. The work focuses on the realization of a biocathode by substituting the traditional, precious and costly Platinum metal catalyst with an enzymatic catalyst system.
Experimental Materials All reagents were procured from Sigma Aldrich. Laccase from Trametes Versicolor (E.C. 1.10.3.2, 24 U/mg) was stored at 20 C. Other chemicals were Monosodium phosphate Disodium phosphate (Na2HPO4), Aniline (NaH2PO4), (C6H5NH2), 2, 2- azino-bis (3-ethylbenzothiazoline-6sulphonic acid) (ABTS), Dimethyl sulfoxide (DMSO), 1-Ethyl3-(3-dimethylaminopropyl) carbodiimide (EDC), Multi-Walled Carbon nanotube carboxylic acid functionalized(MWCNTCOOH, average diameter Length, 9.5 nm 1.5 mm), N-hydroxy succamide (NHS), Hydrochloric acid (HCl) and Acetone. The pencil leads tested were B, H, 3H, and 5H, purchased from a local bookstore, of 60 mm length and varying diameters (2 mm, 1.82 mm, 1.9 mm and 1.88 mm for pencil leads B, H, 3H and 5H respectively). The supporting electrolyte phosphate buffer solution (PBS 0.1 M, pH 5.0) was arranged from NaH2PO4 and Na2HPO4 for biocathode electrochemical analysis. For all the experiments, ultrapure water with resistivity of 18.2 M U cm was used.
Apparatus BET surface area analyzer Smartsorb 93 type surface area analyzer was used to calculate surface area of modified PGEs by dynamic BrunauereEmmetteTeller(BET) principle. The Nitrogen gas used for adsorption and different gas mixture percentages were used to measure total pore volume and surface area. Prior to the measurements, all of the samples were degassed at 473 K for 2 h. The user friendly software stored the data and computed the final surface area and pore volume values from adsorption desorption curves displayed on the screen.
Potentiostat/galvanostat The electrodeposition of PANI and electrochemical analysis were accomplished using a computer controlled Auto lab PGSTAT-302Ntype Potentiostat (Metrohm Auto lab, the Netherlands) driven by NOVA 1.11 software (Eco Chemie, the
Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016
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Netherlands). The experiments were carried out in a conventional three electrode cell (25 ml) under an aerobic condition, composed of working electrode (modified PGEs), Ag/AgCl (3 M KCl) reference electrode and a Platinum wire counter electrode.
Scanning electrode microscope (SEM) The morphological studies of modified PGEs were examined using ZEISS Supra 40VP SUPRA 40 VP Field Emission Scanning Electron Microscope.
Conductivity measurements The conductivity of modified and unmodified pencil leads was calculated using digital multivoltmeter (Mastech MAS830L). The PGEs (bare or modified) were placed in a conducting device which consists of the brass stamp electrodes and the distance between them is maintained consistent by tightening the compression screws. A digital multimeter was used to measure the resistance across the two brass stamp electrodes. The standard equation was used to find the resistivity from resistance values of the modified and unmodified PGEs. Resistivity (r) ¼ R.A/d where A is the effective area of the measuring electrode and d is the distance between two measuring electrodes. Inverse of resistivity is the conductivity.
Procedure Pretreatment of pencil graphite electrodes (PGE) The wooden part of pencils was carefully peeled out with a razor blade. The obtained pencil lead rods were cleaned with 0.5 M HCl solution in ultra-sonication bath for 2 min. Subsequently, they were washed and sonicated in sequence with the deionized water for few times and in acetone for a couple of minutes respectively, and kept for drying in a lab oven for 2 h at 90 C.
Electropolymerization of aniline Pretreated PGEs were used to perform electropolymerization of aniline using cyclic voltammetry (CV) in the range 0.2 V to 1 V at 50 mV/s scanrate. To prepare the electrolyte solution for aniline electropolymerization, 10 ml of aniline was dissolved in the solution of 100 ml of 1.0 M HCl. To get rid of surplus unbound PANI, after polymerization, the PGEs were washed with deionized water. The electrodeposition of PANI on PGE involved the standard steps - adsorption, nucleation, and polymerization.
Preparation of PGE/MWCNT (BC1) electrode Deposition of MWCNT on PGE surface was done by dip coating. The MWCNT-COOH (originally purchased as 1 mg/ml) was isolated in DMSO solvent by ultra-sonication for 2 min. Each pretreated bare PGE was immersed in prepared MWCNTCOOH dispersion for complete wetting and to get sufficient interaction of PGEs with MWCNT solution. A film of MWCNTCOOH deposits on surface of PGEs. Thereafter, the samples were kept in hot air oven (2 h at 90 C) to evaporate solvent
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from their surface. This procedure was repeated 4 times to deposit adequate amount of MWCNT on PGE surface.
Preparation of PGE/PANI/MWCNT (BC2) electrode First, 30 mM EDC and 90 mM NHS were dissolved in PBS, and then MWCNT solution was prepared (MWCNT in EDC/NHS in 1:1 ratio) and the PANI coated PGEs were immersed into MWCNT solution for 2 h. Thereafter, by bioconjugate technique, the carboxylated MWCNT were covalently attached to the free amine group of PANI by amide bond. EDC/NHS compounds were used to activate carboxylic group in organic solvents.
Fabrication of biocathode from modified PGEs First, Laccase enzyme solution was prepared by mixing 3 mg/ml Laccase in 0.1 M PBS. Biocathode were developed by dispersing PGE/MWCNT (BC1) and PGE/PANI/MWCNT(BC2) modified electrodes into the Laccase enzyme solution and letting it remain at room temperature for 24 h. The enzyme was covalently attached to the activated carboxylic group of MWCNT by amide bond on modified electrodes. Later, fabricated biocathodes were washed with PBS to washout unbound enzymes.
Electrochemical measurements Electrochemical characterization of unmodified (bare) and modified PGEs was performed by open circuit potential (OCP) and cyclic voltammetry (CV) techniques. The supporting electrolyte to conduct these tests was PBS (0.1 M, pH 5.0) with 1 mM which ABTS as cathodic mediator [52]. MET is preferred over DET in this study as only a few enzymes are capable of DET [10,53]. All measurements were carried out with a scan rate of 50 mV/s at the room temperature in air saturated buffer solution.
Storage of Laccase immobilized biocathode The fabricated biocathode immobilized with Laccase enzyme on modified PGEs were stored in PBS at 4 C.
Results and discussions Surface characteristics of modified PGEs Scanning electron microscopy (SEM) The surface morphology of pencil graphite electrodes fabricated from 5H pencil leads were characterized by Scanning Electron Microscopy at each step of the fabrication. Fig. 1 shows the SEM image of bare PGE at 5 and 100 magnifications, clearly indicating smooth, layered and featureless morphology with some irregular surface depressions. Fig. 2a shows the SEM image of PGE modified with MWCNT by dip coating. It shows non-uniform distribution of nanotubes developed by their intermolecular interaction and mechanical linking. They appear to be arranged in the form of wires or packets with close packed stacking through van der waals force. Fig. 2b shows SEM image of the surface of PGE modified
Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016
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Fig. 1 e SEM images of pretreated unmodified PGEs from 5H pencil at varying resolutions (a) 5£ (b) 100£.
Fig. 2 e SEM images of (a) PGE/MWCNT (BC1) (b) PGE/PANI/MWCNT (BC2) electrodes for 5H pencil.
with PANI/MWCNT. As can be seen, even though the surface morphology was similar, the MWCNT attached to the nanoporous background PANI by covalent bond shows the presence of thick dense matrix of nanotubes. This offers more free carboxylic groups for high loading of enzymes on to the
surface by amide bond. In Fig. 3a, the SEM images for Laccase immobilized on the PGE coated with MWCNT show the formation of enzyme agglomerates. Although the dispersion of nanotubes on PGE increases the surface area, the random and non-uniform dispersion of nanotubes on the surface of 5H PGE
Fig. 3 e SEM images of (a) PGE/MWCNT (BC1), (b) PGE/PANI/MWCNT (BC2) electrode with immobilized Laccase enzyme for 5H pencil. Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016
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resulted in the ineffective dispersion of the enzymes. Fig. 3b, where Laccase was immobilized on PG/PANI/MWCNT, shows uniform dispersion of the enzymes. Thus, the modification of the PGE with the conducting materials of PANI and MWCNT resulted in greater immobilization of Laccase on the electrode and increased the availability of the enzymes for catalytic activity. Although the PGE was optimized by coating it with conducting material, the chemical and structural properties of various pencil leads also contribute to the electrode properties. The diverse morphological characteristics of various leads were due to their varying composition in terms of graphite content, polymers and clay content [Table 1]. PGEs fabricated from various grades of pencil leads were therefore studied and the electrode properties were found to be optimal for 5H PGE for which the SEM results have been discussed in detail.
Conductivity measurement Electrical conductivity of modified and unmodified(bare) PGEs was measured at room temperature and results are summarized in Table 1. In case of bare PGEs, the conductivity is found to be increasing with increasing graphite percentage of Pencil leads. The highest conductivity recorded for B pencil can be attributed to the highest content of graphite (71%) (See Table 1). The PGEs coated with MWCNT showed increased electrical conductivity upon bare PGEs due to the conducting nature of MWCNT [54]. However, PGEs upon coating with PANI and MWCNT resulted in lower conductivity than that of bare PGE and MWCNT coated PGEs. The reduction in conductivity is due to the random and mixed graphite-insulating structure and with the random assembly of graphite particles inside the rigid non-conducting matrix of binding material (clay and wax).
BET surface area and pore volume measurement The specific surface area (SBET) and pore volume (SPV) of pencil graphite electrodes fabricated from various grades of pencil leads were calculated at each step of the fabrication. The results are shown in Table 1. The bare 5H PGE showed higher surface area with high pore volume distribution. 5H pencil is the hardest pencil among the tested pencils with high content of clay (42%) causing more disorder with high pore volume distribution and surface area, since clay contributes to increase the disorder in the host layered structure of graphite [29]. Further, very less surface area and Pore volume distribution was observed for PANI coated 5H PGE confirming the
formation of thick film of conducting polymer on the surface. The pore volume and surface area for tested pencils (H, 3H, 5H, and B) are shown in Fig. 4.
Electrochemical characterization of PGE The electrochemical performance of unmodified (bare) and modified (PGE/MWCNT/Lac, PGE/PANI/MWCNT/Lac) electrodes were examined. In order to characterize the fabricated biocathode, Open circuit potential (OCP) and Cyclic voltammetry (CV) based analysis were carried out for oxygen reduction reaction.
Open circuit potential (OCP) The OCP of fabricated biocathodes from various grades of pencil leads were measured in air saturated PBS with Ag/AgCl as reference electrode, Platinum wire as counter electrode. Table 2 summarizes the OCP values calculated for 300 s. The measured values were very close to the reduction potential of dioxygen. From the results, it was observed that among the tested pencils, B pencil showed highest OCP for both bare and modified electrodes owing to the higher graphite content (Table 1). Slight reduction in OCP was observed for the same pencil when coated with MWCNT and PANI/MWCNT. This may be due to the blocking of MWCNT with non-conducting binder material used for manufacturing of pencils and also the varying structural properties of PGE surfaces. Further, lower OCP was observed for bare 5H pencil without any modification due to its less graphite content (Table 1). Subsequently, the pencil leads were modified with conducting material MWCNT and PANI. With BET surface area measurements (Table 1), it was observed that 5H pencil possessed highest surface area and pore volume distribution, which facilitated more amount of MWCNT on the surface of 5H pencil. Such modification led to high electron transfer rate, attributed to large OCP compared to unmodified PGE. Comparative graphs for all pencils are shown in Fig. 5 for unmodified (bare) and modified PGEs for all the four pencils.
Cyclic voltammetry (CV) Cyclic Voltammetry of bare and modified (PGE/MWCNT/Lac (BC1), PGE/PANI/MWCNT/Lac (BC2))electrodes was recorded in the voltage range 1.0 V to 1.0 V at 50 mV/s scan rate [52] and substantial difference was observed in their response The obtained current densities calculated from geometrical surface area of pencil leads are tabulated in Table 2. All the
Table 1 e Surface characteristics of unmodified (bare) and modified PGEs. S. no
1 2 3 4 *
Pencil type
Graphite (%)
Clay (%)
H 3H 5H B
63 58 52 71
31% 36% 42% 23%
SBET(m2/gr)
SPV (cm3/gr)
Conductivity (S/cm)
bare
BC1
BC2
bare
BC1
BC2
bare
BC1
BC2
2.23 3.67 5.63 3.15
2.13 2.86 6.26 3.87
0.01 0.08 1.79 3.02
0.0034 0.0024 0.0093 0.0046
0.0017 0.0032 0.0074 0.0056
0.0001 0.0002 0.0045 0.0042
876 922 390 121247
1071 1090 405 142784
1045 653 401 445
BC1 e PGE coated with MWCNT *BC2 —PGE coated with PANI/MWCNT.
Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016
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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 x x x ( 2 0 1 7 ) 1 e1 0
Fig. 4 e Comparative graphs of (a) pore volume (SPV) and (b) surface area (SBET) of all tested pencils.
modified PGEs showed well defined voltammetric peaks at a peak potential close to the redox potential of T1 copper active site determined for Trametes Versicolor Laccase where the ABTS mediator oxidized and transferred electrons to trinuclear copper center T2/T3 of Laccase enzymes. The fully reduced Laccase enzyme reacted with dioxygen and converted into water [51,55]. Thus confirming the catalytic character of Laccase enzyme towards the O2/H2O redox couple (see Fig. 6). From CV studies, the low peak currents observed for bare PGEs are indicative of minimal reduction reaction occurring in the absence of Laccase catalyst. Further, 5H PGE which has the least graphite content among tested pencil leads has
registered least response towards electrochemical reaction. The CV of BC2 (Fig. 8) electrode exhibited enhanced current response than BC1 (Fig. 7) and bare PGE. As mentioned in Table 1, for 5H BC2 (with PANI/MWCNT), the surface area and pore volume distribution reduced (in comparison to bare pencil and BC1). This confirmed the electrochemical deposition of thick layer of highly conducting Emeraldine salt form of polyaniline (PANI) film on electrode surfaces [56]. Such scheme has dual advantage e first, it supports for high peak currents due to the enhanced overall conductivity, and second, the composite layer ensures direct electron transfer (DET) process to the modified electrode surfaces along with ABTS cathodic mediator.
Table 2 e Electrochemical Characteristics of unmodified (bare) and modified PGEs. S. No
1 2 3 4
Pencil type
H 3H 5H B
Current density (mA/cm2)
OCP (vs. Ag/AgCl) (V) Bare
BC1
BC2
Bare
BC1
BC2
0.477 0.516 0.467 0.578
0.571 0.536 0.574 0.517
0.583 0.573 0.528 0.564
51.724 66.260. 8.372 33.616
15.138 11.862 62.790 70.771
436.140 231.912 1209.236 503.263
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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 x x x ( 2 0 1 7 ) 1 e1 0
Fig. 5 e Comparative studies of obtained OCP values for four (H, 3H, 5H and B) pencils unmodified (bare) and modified.
From comparative studies, 5H pencil modified with PANI/MWCNT showed highest current density 1209 mA/ cm2. The highest current response of 5H pencil attribute to the high carboxylic groups on surface with MWCNT as confirmed from SEM images. Table 2 shows the peak currents for all the PGEs, studied in this work, both
unmodified and modified. Observation of overall trend on obtained results for various PGEs, the varying amount of graphite content in pencil leads and composition of the binding material like clay and pencil hardness are the main cause of the difference in response characteristics of PGEs.
600 500
5H
3H
H
B
400 300
Current(μA)
200 100 0 -100 -200 -300 -400 -500 -0.2
0
0.2
0.4 0.6 Applied Potential (V)
0.8
1
1.2
Fig. 6 e Cyclic voltammetry of unmodified (bare) PGEs (H, 3H, 5H and B).
Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016
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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 x x x ( 2 0 1 7 ) 1 e1 0
2000 B
5H
3H
H
1500
Current(μA)
1000
500
0
-500
-1000
-1500 -0.4
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
Applied Voltage(V) Fig. 7 e Cyclic voltammetry of PGE/MWCNT (BC1) electrode immobilized with Laccase enzyme for four pencils (H, 3H, 5H and B).
Fig. 8 e Cyclic voltammetry of PGE/PANI/MWCNT (BC2) electrode immobilized with Laccase enzyme for four pencils (H, 3H, 5H and B). Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016
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 x x x ( 2 0 1 7 ) 1 e1 0
Conclusions Cost effective biocathodes, PGE/MWCNT (BC1) and PGE/PANI/ MWCNT (BC2) immobilized with Laccase enzymes, were fabricated by screening various readily available grades of pencil leads with different carbon content. Morphological characterization confirmed micro-and-nanoporous structures proving uniform distribution of the immobilized Laccase enzyme. While various tested pencils exhibited different response characteristics towards oxygen reduction reaction, any trend among the pencils with respect to graphite was not experiential. However, the electrochemical behavior of the pencil graphite electrode (PGEs) was found to be greatly influenced by the different compositions, such as clay and other binder components, leading to their disordered structure and pencil hardness. CV and OCP studies indicate that 5H pencil coated with PANI and MWCNT have highest potential for use as biocathode in EBFC.
Acknowledgement The work was financially supported from the India-Korea Project by the Department of Science and Technology, Government of India (INT/Korea/P-18/2013) to SG, the Research Initiation Grant from BITS-Pilani, Hyderabad Campus to SG, and the Nanomission project by the Department of Science and Technology, Government of India (SR/NM/NS-82/2016 (G)) to PKD.
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Please cite this article in press as: Bandapati M, et al., Screening various pencil leads coated with MWCNT and PANI as enzymatic biofuel cell biocathode, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.016