Accepted Manuscript Title: Enzyme Adsorption, Precipitation and Crosslinking of Glucose Oxidase and Laccase on Polyaniline Nanofibers for Highly Stable Enzymatic Biofuel Cells Author: Ryang Eun Kim Sung-Gil Hong Su Ha Jungbae Kim PII: DOI: Reference:
S0141-0229(14)00138-0 http://dx.doi.org/doi:10.1016/j.enzmictec.2014.08.001 EMT 8666
To appear in:
Enzyme and Microbial Technology
Received date: Accepted date:
4-8-2014 6-8-2014
Please cite this article as: Kim RE, Hong S-G, Ha S, Kim J, Enzyme Adsorption, Precipitation and Crosslinking of Glucose Oxidase and Laccase on Polyaniline Nanofibers for Highly Stable Enzymatic Biofuel Cells, Enzyme and Microbial Technology (2014), http://dx.doi.org/10.1016/j.enzmictec.2014.08.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights (for review)
Highlight Enzyme adsorption, precipitation and crosslinking (EAPC) approach offered high loading and stability of enzymes. Enzymatic biofuel cells were successfully fabricated and operated using enzyme anode
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(glucose oxidase) and cathode (laccase).
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Enzymatic biofuel cells using EAPC-based electrodes improved both power density output
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*Manuscript
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Enzyme Adsorption, Precipitation and Crosslinking of Glucose
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Oxidase and Laccase on Polyaniline Nanofibers for Highly Stable
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Enzymatic Biofuel Cells
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Ryang Eun Kim1,†, Sung-Gil Hong 1,†, Su Ha2,*, Jungbae Kim1,**
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Republic of Korea
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Department of Chemical and Biological Engineering, Korea University, Seoul 136-701,
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WA 99164, USA
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School of Chemical Engineering and Bioengineering, Washington State University, Pullman,
These authors contributed equally to this work.
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Corresponding Authors
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* Prof. Su Ha
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The Gene and Linda Voiland School of Chemical Engineering and Bioengineering,
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Washington State University
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Pullman, WA 99164, USA
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Tel.: 509 335 3786
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Fax: 509 335 4806
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E-mail address:
[email protected] us
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** Prof. Jungbae Kim
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Department of Chemical and Biological Engineering,
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Korea University
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Seoul 136-701, Republic of Korea
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Tel.:+82 2 958 4850
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Fax: +82 2 926 6102
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E-mail address:
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Abstract Enzymatic biofuel cells have many great features as a small power source for medical,
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environmental and military applications. Both glucose oxidase (GOx) and laccase (LAC) are
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widely used anode and cathode enzymes for enzymatic biofuel cells, respectively. In this paper,
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we employed three different approaches to immobilize GOx and LAC on polyaniline nanofibers
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(PANFs): enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC) and enzyme
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adsorption, precipitation and crosslinking (EAPC) approaches. The activity of EAPC-LAC was
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32 and 25 times higher than that of EA-LAC and EAC-LAC, respectively. The half-life of
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EAPC-LAC was 53 days, while those of EA-LAC and EAC-LAC were 6 and 21 days,
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respectively. Similar to LAC, EAPC-GOx also showed higher activity and stability than EA-
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GOx and EAC-GOx. For the biofuel cell application, EAPC-GOx and EAPC-LAC were applied
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over the carbon papers to form enzyme anode and cathode, respectively. In order to improve the
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power density output of enzymatic biofuel cell, 1,4-benzoquinone (BQ) and 2,2′-azino-bis(3-
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ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were introduced as the electron
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transfer mediators on the enzyme anode and enzyme cathode, respectively. BQ- and ABTS-
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mediated enzymatic biofuel cells fabricated by EAPC-GOx and EAPC-LAC showed the
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maximum power density output of 37.4 μW/cm2, while the power density output of 3.1 μW/cm2
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was shown without mediators. Under room temperature and 4 °C for 28 days, enzymatic biofuel
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cells maintained 54 and 70 % of its initial power density, respectively.
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Keywords: Enzyme adsorption, precipitation and crosslinking (EAPC); Polyaniline nanofibers;
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Glucose oxidase; Laccase; Enzymatic biofuel cells
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1.
Introduction Enzymatic biofuel cells are energy conversion devices that could efficiently convert the
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chemical energy of biofuels into electrical energy using enzymes as biocatalysts [1]. They can
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operate under mild condition such as a neutral pH and an ambient temperature [1,2]. Enzymatic
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biofuel cells have a great potential to be used as a portable and uninterrupted power source for
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the various medical, environmental and military applications by using the fuels such as glucose,
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which are commonly available to biological and environmental systems [3-7]. However, despite
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of promising application of enzymatic biofuel cells, their low power density and short lifetime,
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both of which linked to low loading and poor stability of enzymes, have been identified as two
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critical issues that need to be addressed [8]. As a potential solution, nanobiocatalytic approaches,
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in which enzyme are incorporated into nanostructured materials, have been employed to provide
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enhanced the loading and stability of enzymes [9]. In particular, polyaniline nanofibers (PANFs)
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is a very interesting supporting material because they can offer a large surface area with
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nanofiber matrices as well as high electron conductive property [10]. Moreover, PANFs can be
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easily and economically synthesized when compared to other nanostructured materials such as
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electrospun nanofibers, nanoparticles, carbon nanotubes and mesoporous materials. Because of
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these promising properties, PANFs have been employed to immobilize and stabilize various
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enzymes on PANFs [11-13].
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In the present study, we immobilized glucose oxidase (GOx) and laccase (LAC) on PANFs
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via the enzyme adsorption, precipitation and crosslinking (EAPC) approach, together with the
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enzyme adsorption (EA) and enzyme adsorption and crosslinking (EAC) approaches as controls,
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to fabricate enzymatic biofuel cells. The anode is consisted of GOx immobilized in the form of
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EAPC (i.e., EAPC-GOx), while the cathode is consisted of LAC immobilized in the form of 4
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EAPC (i.e., EAPC-LAC). We investigated the effect of mediators on each electrode, and
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evaluated their biofuel cell performances in terms of power density and long-term stability by
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using glucose as the fuel. Based on our knowledge, it is first time to fabricate and successfully
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operate enzymatic biofuel cells by utilizing both the enzyme anode and enzyme cathode in the
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form of EAPC.
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2.
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2.1. Materials
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Materials and methods
Laccase (LAC) from Trametes versicolor, glucose oxidase (GOx) from Aspergillus niger, syringaldazine,
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tetramethylbenzidine (TMB), glutaraldehyde solution (GA, 25%), ammonium sulfate, 2,2’-
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azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,4-benzoquinone
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(BQ), Nafion® solution (5 wt%), aniline and ammonium persulfate were purchased from Sigma
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(St. Louis, MO, USA). Carbon papers (CPs) and Nafion® 117 membrane were purchase from
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Fuel Cell Store (Boulder, CO, USA).
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2.2. Synthesis of polyaniline nanofibers
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β-D-glucose,
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methanol,
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Polyaniline nanofiber was synthesized by initiating polymerization of aniline in acidic
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condition using ammonium persulfate as an initiator [10]. First, 9 M aniline monomer solution
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and 0.1 M ammonium persulfate solution were prepared in 1 M HCl. Both aniline and
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ammonium persulfate solutions in HCl were mixed and shaken using 200 rpm at room
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temperature for 24 hrs. After a completion of the polymerization reaction, PANFs were
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centrifuged down, washed using DI water excessively for 3 times, suspended in DI water and stored at 4 ˚C until use.
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2.3. Immobilization of LAC and GOx on PANFs
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PANFs were used for the immobilization of LAC and GOx in three different enzyme
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immobilization methods: EA, EAC, and EAPC. PANFs were washed with 100 mM phosphate
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buffer (PB) solution (pH 7.0) for 3 times prior to the immobilization processes. Immobilized
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LAC in the form of enzyme adsorption on PANF (i.e., EA-LAC) was prepared by mixing the 2
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mg of PANF with the LAC solution (10 mg/mL) in 100 mM PB (pH 6.5) under the shaking
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condition at 150 rpm for 1 hr. For the preparation of immobilized LAC in the form of enzyme
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adsorption and crosslinking on PANF (i.e., EAC-LAC), the glutaraldehyde (GA) as the chemical
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crosslinking agent was introduced to make a final concentration of 0.5% (w/v) to the EA-LAC
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sample under the shaking condition at 50 rpm and 4 ˚C for 17 hrs. To prepare the immobilized
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LAC in the form of enzyme adsorption, precipitation and crosslinking on PANF (i.e., EAPC-
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LAC), the ammonium sulfate solution was introduced into the 100 mM PB solution (pH 6.5)
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containing both LAC and PANF to make a concentration of 50% (w/v). In the presence of the
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ammonium sulfate salt, the free LAC (i.e., LAC that is not adsorbed over PANF surface) was
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precipitated out to form the enzyme aggregates. After shaking at 200 rpm for 30 mins, the GA
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solution was added into the mixture to make a concentration of 0.5% (w/v) to chemically
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crosslink the precipitated LAC aggregates over the surface of PANF at 4 ˚C for 17 hrs. To cap
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un-reacted aldehyde groups, the samples were shaken at 200 rpm in 100 mM Tris-HCl buffer
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(pH 7.4) solution for 30 min and the samples were excessively washed for 3 times with the 100
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mM PB solution (pH 6.5). EA-LAC, EAC-LAC and EAPC-LAC were stored in 100 mM PB
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solution (pH 6.5) at 4 ˚C until use. The EA-GOx, EAC-GOx and EAPC-GOx were also prepared
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by following the same protocols that were used for the immobilization of LAC on PANF as
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described above.
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2.4. Activity and stability measurement of immobilized LAC and GOx on PANFs
The activity was calculated from the time-dependent change of absorbance, and the stabilities
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of samples were checked by measuring the residual activity time-dependently after incubation in
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buffer solution at room temperature. The measurement of LAC activity was based on the
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oxidation of syringaldazine [14]. Syringaldazine (7.8 mg) dissolved in methanol (10 ml) with a
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final concentration of 0.216 mM. 100 μL of the solution containing the immobilized LAC on
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PANFs (0.1 mg/ml) was mixed with 800 μL of 100 mM PB solution (pH 6.5) and the mixtures
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were heated at 30 ˚C for 10 mins. This heated mixture was added with 100 μL of syringaldazine
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solution (0.216 mM) and the absorbance at 530 nm (A530) was measured by using UV
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spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan).
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The activity of immobilized GOx on PANFs was measured by GOx assay [15]. The
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measurement of activity needs a reaction cocktail containing TMB and glucose solution.
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Reaction cocktail was made of 12 ml of TMB solution (0.576 mg/ml) and 2.5 ml glucose
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solution (110.1 mg/ml). To measure activity of immobilized GOx, 890 μL of reaction cocktail
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was mixed with 10 μL HRP solution (3.798 mg/ml). Then, 100 μL of the solution containing the
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immobilized GOx on PANFs (1 μg/ml) was added to 900 μL of the mixed solution. The
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absorbance of immobilized GOx was measured at 655 nm (A655) by using UV spectrophotometer.
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2.5. Preparation of enzyme electrodes 7
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Carbon papers (CPs, thickness of 370 μm, 0.44 g/cm3) were treated with acid before use. In
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a typical preparation, 2cm × 2cm squares of CPs was added to an acid solution composed of
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H2SO4 (98%, 30 ml) and HNO3 (70%, 10 ml) for overnight at room temperature under a stirring
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condition. Then, acid-treated CPs were washed with distilled water, dried at vacuum condition
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and stored at room temperature until use. To prepare the GOx-based anode, the immobilized
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GOx on PANF sample was mixed with Nafion® solution (final conc. 0.5 wt %) and this mixture
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was stored at 4 ˚C for 1 hr. Acid-treated CPs (0.332 cm2) was soaked into the mixture for 10
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mins, followed by drying at ambient conditions. After drying, the prepared GOx-based enzyme
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anode was stored in 100 mM PB solution (pH 7.0) at 4 ˚C. For the LAC-based cathode, ABTS
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(30 mM, 3.3 mg) was added to the mixture of Nafion® and immobilized LAC on PANF sample.
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When the LAC-based cathode containing ABTS was dried, it was stored in 100 mM PB solution
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(pH 7.0) at 4 ˚C. Since ABTS has high solubility in aqueous solution, washing was not carried
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out [16].
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2.6. Biofuel cell operation measurement
The electrochemical measurements were performed by using Bio-Logic SP-150 (Knoxville,
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TN, USA). The performance of enzymatic biofuel cells was measured by circulating 200 mM
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glucose solution with and without 10 mM BQ in 100 mM PB solution (pH 7.0) at the flow rate of
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0.4 ml/min within the GOx-based anode. For the LAC-based cathode, the air-breathing structure
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was used to utilize the ambient air as its oxygen source. The Bio-Logic SP-150 was used to
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measure the current and voltage outputs of biofuel cell by 3 minute interval under the various
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load conditions. The power density (μW/cm2) was calculated by multiplying current and voltage
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and then divided by surface of electrode (0.332 cm2).
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3.
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3.1. Immobilization of LAC and GOx on PANFs
Results and discussion
Figure 1 shows schematic illustrations for the immobilization of enzymes (LAC and GOx)
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in three different enzyme immobilization methods: EA, EAC, and EAPC. The scanning electron
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microscope (SEM) images of PANFs, EA, EAC and EAPC were shown in Figure 2 for both
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GOx and LAC samples. The nanofiber morphology of EA and EAC samples was fairly similar
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with pristine PANFs (SEM image of pristine PANFs is not shown), whereas EAPC showed
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remarkably thicker nanofibers revealing the enzyme coating layer over the surface of PANFs. By
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checking twenty samples of nanofiber images, the average thicknesses of EA-LAC, EAC-LAC
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and EAPC-LAC were estimated to be 61 ± 6, 82 ± 7 and 115 ± 6 nm, respectively, while those of
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EA-GOx, EAC-GOx and EAPC-GOx were 75 ± 5, 91 ± 7 and 142 ± 15 nm, respectively. The
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thickness of the enzyme coating layer for EAPC samples increased significantly than those of EA
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and EAC samples due to their improved enzyme loading induced by the ammonium sulfate
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assisted enzyme precipitation step to form the enzyme aggregates and its subsequent chemical
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crosslinking step. Since the only difference between EAC and EAPC samples was the addition of
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the enzyme precipitation process for EAPC sample, the SEM data clearly indicates that the
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enzyme precipitation process is a critical step in order to form the thick enzyme-coating layer
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over the supporting material. Furthermore, it is interesting to note that EAPC samples with GOx
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offer a much thicker enzyme coating layer than that of EAPC samples with LAC. GOx can be
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crosslinked more rigorously than LAC because the number of lysine residues per each GOx
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molecule is 15 [17], while each LAC molecule has only 8 lysine residues [18].
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3.2. Activity and stability of immobilized LAC and GOx on PANFs 9
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Activity of immobilized LAC and GOx on PANFs is shown in Figure 3. The activities of
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EA-LAC, EAC-LAC and EAPC-LAC samples were 1.9, 2.4 and 61.4 A530/min per mg of PANFs,
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respectively (Fig. 3a). The activity of EAPC-LAC sample was 32 and 25 times higher than that
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of EA-LAC and EAC-LAC samples, respectively. The activities of immobilized GOx on PANFs
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have similar tendencies (Fig. 3b). The activities of EA-GOx, EAC-GOx and EAPC-GOx were
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35.9, 124.6 and 5930 A655/min per mg of PANFs. EAPC-GOx approach offers 165 and 47 times
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higher activity than that of EA-GOx and EAC-GOx approaches, respectively. The higher activity
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of EAPC than that of EA and EAC can be interpreted in terms of enhanced enzyme loading by
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the combination of the precipitation and crosslinking processes. These results matched well with
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morphological changes of EA, EAC and EAPC samples shown in their corresponding SEM
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images (Figure 2).
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Figure 4 shows the stabilities of EA-LAC, EAC-LAC and EAPC-LAC over the 78 days at
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room temperature. After 78 days, EAPC-LAC maintained 43% of the initial activity, whereas
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EA-LAC and EAC-LAC maintained 5% and 12% of their initial activities, respectively. The
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inactivation profiles of all samples were bi-phasic with faster inactivation followed by the slower
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inactivation at the later phase. The faster inactivation in the early phase can be explained by the
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labile form of enzymes after being immobilized. The half-life of each sample in the early phase
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was estimated from the first-order inactivation kinetics. The estimated half-lives of EA-LAC,
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EAC-LAC and EAPC-LAC were 6, 21 and 53 days, respectively. The stability of immobilized
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GOx on PANFs was measured in our previous work [12]. After 56 days, the relative activities of
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EA-GOx, EAC-GOx and EAPC-GOx samples were 22%, 19% and 91%. EA and EAC
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approaches resulted in poor enzyme stability due to the denaturation and continuous leaching of
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enzymes from PANFs. Furthermore, the higher enzyme stability offered by EAPC approach can
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be explained in terms of its effective enzyme precipitation and crosslinking steps on PANFs.
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Precipitation by ammonium sulfate allows enzyme molecules to be closely packed. This closely
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packed and large enzyme aggregates can make the formation of multi-point chemical linkages on
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the surface of each enzyme molecule more effective when they are treated with glutaric
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dialdehyde (glutaraldehyde, GA) for the crosslinking of enzymes. These multi-point chemical
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linkages on the enzyme surface can effectively prevent denaturation and leaching of enzymes,
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thus stabilizing the activity of enzymes over the long operation time as it was demonstrated by
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our EAPC samples [19,20].
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3.3. Enzymatic biofuel cells
Figure 5 shows the schematic of enzymatic biofuel cell. Based on the activity and stability
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tests, both GOx and LAC showed the best performances when they were immobilized in the
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form of EAPC. Thus, we fabricated the enzyme anode and enzyme cathode by entrapping
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EAPC-GOx and EAPC-LAC over the carbon paper using Nafion® as a binder, respectively. As
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indicated in Figure 5, the enzymatic biofuel cell is made of the anode chamber, anode current
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collector, enzyme anode, proton exchange membrane (Nafion® 117), enzyme cathode, cathode
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current collector and cathode holder. During the cell operation, the glucose fuel was
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electrochemically oxidized by GOx to produce two electrons, two protons and by-product (e.g.
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gluconolactone) over the enzyme anode. Generated electrons from the anode flow to the cathode
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through the external load circuit to provide the electrical power. At the cathode, both electrons
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and protons are combined together to form H2O.
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Figure 6a and Figure 6b show the voltage-current (V-I) curves and the power density-
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current (P-I) curves of enzymatic biofuel cell with EAPC-GOx and EAPC-LAC at room 11
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temperature, respectively. Without any mediator, the open circuit voltage (OCV) is about 0.34 V
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and the maximum power density is 3.1 μW/cm2. According to previous work [12], we
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demonstrated that the power density output depends on the enzyme loading. As the enzyme
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loading increases per unit weight of PANFs, the enzyme activity also proportionally increases
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per unit weight of PANFs up to a certain value of enzyme loading. For the anode case, the
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enzyme activity is closely related with electron generation rate and it represents the maximum
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amount of electrons that can be generated without considering the charge transfer resistance. We
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speculate that the large amount of electrons generated per unit time by the high enzyme loading
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of EAPC-based electrode increases the probability of collecting these electrons at the current
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collector. However, to significantly increase the power density output of our enzymatic biofuel
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cell, it is also important to improve the electron transfer rate of its electrodes because only a
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limited amount of electrons would be collected if the enzyme electrodes have poor electron
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transfer rates regardless of the enzyme activities.
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In order to increase the electron transfer rates of both enzyme electrodes, the mediators have
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been utilized [2,21,22]. For the present study, BQ and ABTS were used as the electron transfer
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mediators for the enzyme anode and enzyme cathode, respectively [23-25]. BQ is a liquid
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mediator, and it is mixed with the 200 mM of glucose fuel, while ABTS is a solid mediator
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where it is simultaneously entrapped with EAPC-LAC samples using Nafion® binder over the
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carbon paper surface. The performance of biofuel cells containing EAPC-GOx and EAPC-LAC
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with mediators is also shown in Figures 6. According to the voltage-current (V-I) curves of
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enzymatic biofuel cells shown in Figure 6a, when the ABTS is incorporated to the enzyme
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cathode containing EAPC-LAC, the OCV increases from 0.34 to 0.50 V. However, its current
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density rapidly drops in a similar manner as the cell without the ABTS-mediated cathode. On the
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other hand, when both the BQ and ABTS are incorporated to the enzyme anode and enzyme
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cathode, respectively, the enzymatic biofuel cell shows a smaller cell voltage drop as the current
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density increases compared to that of the cell with just ABTS-mediated cathode. This result
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suggests that the overall cell performance is mainly limited by the slower electron transfer rate of
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EAPC-GOx-based anode. Due to this smaller overpotential, the enzymatic biofuel cell with both
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BQ- and ABTS-mediated electrodes generates a much higher current density output as shown in
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Figure 6a.
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Figure 6b shows the power density-current (P-I) curves of enzymatic biofuel cells. The
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power density output of the biofuel cell with just ABTS-mediated cathode does not improve
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much compared to the biofuel cells without the mediators. Consequently, the maximum power
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density outputs of the biofuel cells with no mediators and with just ABTS-mediated cathode are
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3.1 and 5.7 μW/cm2, respectively. However, the biofuel cells with BQ- and ABTS-mediated
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electrodes containing EAPC-GOx and EAPC-LAC showed the maximum power density up to
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37.4 μW/cm2 due to its decreased overpotential mainly offered by the improved electron transfer
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rate of the anode in the presence of the mediator.
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3.4. Performance stability of enzymatic biofuel cells
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To confirm the performance stability of enzymatic biofuel cells with EAPC-GOx and
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EAPC-LAC electrodes, the enzyme electrodes containing EAPC-GOx and EAPC-LAC were
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incubated in the aqueous buffer under two different temperatures (room temperature and 4 ˚C)
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over 28 days (Fig. 7). In a 7-day interval, these enzyme electrodes were taken out from the
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incubation and integrated into our biofuel cell shown in Figure 5 to measure its maximum power
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density using 200 mM glucose solution at ambient temperature. After the biofuel cell test, both 13
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EAPC-GOx and EAPC-LAC electrodes were removed from the cell and washed with 100 mM
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PB solution (pH 7.0 for the anode and pH 6.5 for the cathode) to remove any residue glucose
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solution before they were placed back into the incubation. When incubated over 28 days, the
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maximum power density output of the biofuel cell with the electrodes stored at room temperature
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maintained 54% of its initial value, while the biofuel cell with the electrodes stored at 4 ˚C
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maintained 70% of its initial value. As seen in Figure 7, there is almost no difference in the
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maximum power density of biofuel cell between two different temperatures used for the thermal
290
stability tests within the 7 days. However, as the thermal stability test continues beyond 7 days,
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the power density output of the biofuel cell with the electrodes stored at 4 ˚C shows a lower
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deactivation rate than the biofuel cell with the electrodes stored at room temperature. Overall, the
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biofuel cell with EAPC-GOx and EAPC-LAC electrodes shows a good performance stability,
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which agrees with their stability results shown in Figure 3.
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4.
Conclusions
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In this study, we introduced biofuel cells with anode electrode and cathode electrode based
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on immobilized GOx and LAC on PANFs in the form of enzyme adsorption, precipitation and
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crosslinking (EAPC). EAPC approach demonstrates that both the loading and stability of GOx
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and LAC on PANFs can be significantly improved by introducing the enzyme precipitation and
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crosslinking steps for their immobilization processes. By applying EAPC method to the
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enzymatic biofuel cell, we have achieved both high power density output and improved
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performance stability. As shown in successful application to biofuel cells, it is anticipated that
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EAPC method can be utilized for various types of enzyme-based electrochemical applications
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such as biosensors and enzyme logic gates.
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Acknowledgement
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This work was supported by the grant from the Agency for Defense Development (ADD -14-70-
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04-01).
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Figure Captions
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Figure 1. Schematic illustrations for three different enzyme immobilization methods using
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PANFs: enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC) and enzyme
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adsorption, precipitation and crosslinking (EAPC). Magnified figure of EAPC represents
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crosslinking between enzymes molecules.
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Figure 2. SEM images of (a) EA-LAC, (b) EAC-LAC, (c) EAPC-LAC, (d) EA-GOx, (e) EAC-
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GOx and (f) EAPC-GOx. The scale bars in all images correspond to 1 μm.
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Figure 3. The activities of (a) EA-LAC, EAC-LAC and EAPC-LAC, and (b) EA-GOx, EAC-
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GOx and EAPC-GOx.
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Figure 4. Stabilities of EA-LAC, EAC-LAC and EAPC-LAC on PANFs at room temperature.
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Figure 5. Schematic illustrations of biofuel cells consisting of EAPC-GOx (anode electrode) and
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EAPC-LAC (cathode electrode).
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Figure 6. (a) The voltage-current (V-I) curves and (b) the power density-current (P-I) curves of
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enzymatic biofuel cell containing EAPC-GOx and EAPC-LAC with and without mediators (BQ
380
for the anode mediator and ABTS for the cathode mediator).
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Figure 7. Relative maximum power density of biofuel cells containing EAPC-GOx (anode) and
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EAPC-LAC (cathode), which were stored at room temperature and 4 ˚C over 28 days.
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M
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Fig. 1. Schematic illustrations for three different enzyme immobilization methods using PANFs:
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enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC) and enzyme adsorption,
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precipitation and crosslinking (EAPC). Magnified figure of EAPC represents crosslinking
388
between enzymes molecules.
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ip t cr us an M ed ce pt Ac
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Fig. 2. SEM images of (a) EA-LAC, (b) EAC-LAC, (c) EAPC-LAC, (d) EA-GOx, (e) EAC-
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GOx and (f) EAPC-GOx. The scale bars in all images correspond to 1 μm.
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Ac
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Fig. 3. The activities of (a) EA-LAC, EAC-LAC and EAPC-LAC, and (b) EA-GOx, EAC-GOx
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and EAPC-GOx.
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Fig. 4. Stabilities of EA-LAC, EAC-LAC and EAPC-LAC on PANFs at room temperature.
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ip t cr us
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Fig. 5. Schematic illustrations of biofuel cells consisting of EAPC-GOx (anode electrode) and
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EAPC-LAC (cathode electrode).
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ip t cr us an M ed ce pt
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Fig. 6. (a) The voltage-current (V-I) curves and (b) the power density-current (P-I) curves of
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enzymatic biofuel cell containing EAPC-GOx and EAPC-LAC with and without mediators (BQ
404
for the anode mediator and ABTS for the cathode mediator)..
Ac
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ip t cr us
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Fig. 7. Relative maximum power density of biofuel cells containing EAPC-GOx (anode) and
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EAPC-LAC (cathode), which were stored at room temperature and 4 ˚C over 28 days.
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