kim 2014

Accepted Manuscript

Title: Enzyme Adsorption, Precipitation and Crosslinking ofGlucose Oxidase and Laccase on Polyaniline Nanofibers forHighly Stable Enzymatic Biofuel Cells

Author: Ryang Eun Kim Sung-Gil Hong Su Ha Jungbae Kim

PII: S0141-0229(14)00138-0DOI: http://dx.doi.org/doi:10.1016/j.enzmictec.2014.08.001Reference: EMT 8666

To appear in: Enzyme and Microbial Technology

Received date: 4-8-2014Accepted date: 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 PolyanilineNanofibers for Highly Stable Enzymatic Biofuel Cells, Enzyme and MicrobialTechnology (2014), http://dx.doi.org/10.1016/j.enzmictec.2014.08.001This 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 proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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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

(glucose oxidase) and cathode (laccase).

Enzymatic biofuel cells using EAPC-based electrodes improved both power density output

and performance stability.

Highlights (for review)

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1

Enzyme Adsorption, Precipitation and Crosslinking of Glucose 2

Oxidase and Laccase on Polyaniline Nanofibers for Highly Stable 3

Enzymatic Biofuel Cells 4

5

Ryang Eun Kim1,

, Sung-Gil Hong 1,

, Su Ha2,

*, Jungbae Kim1,

** 6

7

1 Department of Chemical and Biological Engineering, Korea University, Seoul 136-701, 8

Republic of Korea 9

10

2 School of Chemical Engineering and Bioengineering, Washington State University, Pullman, 11

WA 99164, USA 12

13

These authors contributed equally to this work. 14

*Manuscript

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Corresponding Authors 15

* Prof. Su Ha 16

The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, 17

Washington State University 18

Pullman, WA 99164, USA 19

Tel.: 509 335 3786 20

Fax: 509 335 4806 21

E-mail address: [email protected] 22

23

** Prof. Jungbae Kim 24

Department of Chemical and Biological Engineering, 25

Korea University 26

Seoul 136-701, Republic of Korea 27

Tel.:+82 2 958 4850 28

Fax: +82 2 926 6102 29

E-mail address: [email protected] 30

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Abstract 32

Enzymatic biofuel cells have many great features as a small power source for medical, 33

environmental and military applications. Both glucose oxidase (GOx) and laccase (LAC) are 34

widely used anode and cathode enzymes for enzymatic biofuel cells, respectively. In this paper, 35

we employed three different approaches to immobilize GOx and LAC on polyaniline nanofibers 36

(PANFs): enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC) and enzyme 37

adsorption, precipitation and crosslinking (EAPC) approaches. The activity of EAPC-LAC was 38

32 and 25 times higher than that of EA-LAC and EAC-LAC, respectively. The half-life of 39

EAPC-LAC was 53 days, while those of EA-LAC and EAC-LAC were 6 and 21 days, 40

respectively. Similar to LAC, EAPC-GOx also showed higher activity and stability than EA-41

GOx and EAC-GOx. For the biofuel cell application, EAPC-GOx and EAPC-LAC were applied 42

over the carbon papers to form enzyme anode and cathode, respectively. In order to improve the 43

power density output of enzymatic biofuel cell, 1,4-benzoquinone (BQ) and 2,2-azino-bis(3-44

ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were introduced as the electron 45

transfer mediators on the enzyme anode and enzyme cathode, respectively. BQ- and ABTS-46

mediated enzymatic biofuel cells fabricated by EAPC-GOx and EAPC-LAC showed the 47

maximum power density output of 37.4 W/cm2, while the power density output of 3.1 W/cm2 48

was shown without mediators. Under room temperature and 4 C for 28 days, enzymatic biofuel 49

cells maintained 54 and 70 % of its initial power density, respectively. 50

51

Keywords: Enzyme adsorption, precipitation and crosslinking (EAPC); Polyaniline nanofibers; 52

Glucose oxidase; Laccase; Enzymatic biofuel cells 53

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1. Introduction 54

Enzymatic biofuel cells are energy conversion devices that could efficiently convert the 55

chemical energy of biofuels into electrical energy using enzymes as biocatalysts [1]. They can 56

operate under mild condition such as a neutral pH and an ambient temperature [1,2]. Enzymatic 57

biofuel cells have a great potential to be used as a portable and uninterrupted power source for 58

the various medical, environmental and military applications by using the fuels such as glucose, 59

which are commonly available to biological and environmental systems [3-7]. However, despite 60

of promising application of enzymatic biofuel cells, their low power density and short lifetime, 61

both of which linked to low loading and poor stability of enzymes, have been identified as two 62

critical issues that need to be addressed [8]. As a potential solution, nanobiocatalytic approaches, 63

in which enzyme are incorporated into nanostructured materials, have been employed to provide 64

enhanced the loading and stability of enzymes [9]. In particular, polyaniline nanofibers (PANFs) 65

is a very interesting supporting material because they can offer a large surface area with 66

nanofiber matrices as well as high electron conductive property [10]. Moreover, PANFs can be 67

easily and economically synthesized when compared to other nanostructured materials such as 68

electrospun nanofibers, nanoparticles, carbon nanotubes and mesoporous materials. Because of 69

these promising properties, PANFs have been employed to immobilize and stabilize various 70

enzymes on PANFs [11-13]. 71

In the present study, we immobilized glucose oxidase (GOx) and laccase (LAC) on PANFs 72

via the enzyme adsorption, precipitation and crosslinking (EAPC) approach, together with the 73

enzyme adsorption (EA) and enzyme adsorption and crosslinking (EAC) approaches as controls, 74

to fabricate enzymatic biofuel cells. The anode is consisted of GOx immobilized in the form of 75

EAPC (i.e., EAPC-GOx), while the cathode is consisted of LAC immobilized in the form of 76

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EAPC (i.e., EAPC-LAC). We investigated the effect of mediators on each electrode, and 77

evaluated their biofuel cell performances in terms of power density and long-term stability by 78

using glucose as the fuel. Based on our knowledge, it is first time to fabricate and successfully 79

operate enzymatic biofuel cells by utilizing both the enzyme anode and enzyme cathode in the 80

form of EAPC. 81

82

2. Materials and methods 83

2.1. Materials 84

Laccase (LAC) from Trametes versicolor, glucose oxidase (GOx) from Aspergillus niger, 85

syringaldazine, methanol, -D-glucose, horseradish peroxidase (HRP), 3,3,5,5-86

tetramethylbenzidine (TMB), glutaraldehyde solution (GA, 25%), ammonium sulfate, 2,2-87

azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,4-benzoquinone 88

(BQ), Nafion solution (5 wt%), aniline and ammonium persulfate were purchased from Sigma 89

(St. Louis, MO, USA). Carbon papers (CPs) and Nafion 117 membrane were purchase from 90

Fuel Cell Store (Boulder, CO, USA). 91

92

2.2. Synthesis of polyaniline nanofibers 93

Polyaniline nanofiber was synthesized by initiating polymerization of aniline in acidic 94

condition using ammonium persulfate as an initiator [10]. First, 9 M aniline monomer solution 95

and 0.1 M ammonium persulfate solution were prepared in 1 M HCl. Both aniline and 96

ammonium persulfate solutions in HCl were mixed and shaken using 200 rpm at room 97

temperature for 24 hrs. After a completion of the polymerization reaction, PANFs were 98

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centrifuged down, washed using DI water excessively for 3 times, suspended in DI water and 99

stored at 4 C until use. 100

101

2.3. Immobilization of LAC and GOx on PANFs 102

PANFs were used for the immobilization of LAC and GOx in three different enzyme 103

immobilization methods: EA, EAC, and EAPC. PANFs were washed with 100 mM phosphate 104

buffer (PB) solution (pH 7.0) for 3 times prior to the immobilization processes. Immobilized 105

LAC in the form of enzyme adsorption on PANF (i.e., EA-LAC) was prepared by mixing the 2 106

mg of PANF with the LAC solution (10 mg/mL) in 100 mM PB (pH 6.5) under the shaking 107

condition at 150 rpm for 1 hr. For the preparation of immobilized LAC in the form of enzyme 108

adsorption and crosslinking on PANF (i.e., EAC-LAC), the glutaraldehyde (GA) as the chemical 109

crosslinking agent was introduced to make a final concentration of 0.5% (w/v) to the EA-LAC 110

sample under the shaking condition at 50 rpm and 4 C for 17 hrs. To prepare the immobilized 111

LAC in the form of enzyme adsorption, precipitation and crosslinking on PANF (i.e., EAPC-112

LAC), the ammonium sulfate solution was introduced into the 100 mM PB solution (pH 6.5) 113

containing both LAC and PANF to make a concentration of 50% (w/v). In the presence of the 114

ammonium sulfate salt, the free LAC (i.e., LAC that is not adsorbed over PANF surface) was 115

precipitated out to form the enzyme aggregates. After shaking at 200 rpm for 30 mins, the GA 116

solution was added into the mixture to make a concentration of 0.5% (w/v) to chemically 117

crosslink the precipitated LAC aggregates over the surface of PANF at 4 C for 17 hrs. To cap 118

un-reacted aldehyde groups, the samples were shaken at 200 rpm in 100 mM Tris-HCl buffer 119

(pH 7.4) solution for 30 min and the samples were excessively washed for 3 times with the 100 120

mM PB solution (pH 6.5). EA-LAC, EAC-LAC and EAPC-LAC were stored in 100 mM PB 121

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solution (pH 6.5) at 4 C until use. The EA-GOx, EAC-GOx and EAPC-GOx were also prepared 122

by following the same protocols that were used for the immobilization of LAC on PANF as 123

described above. 124

125

2.4. Activity and stability measurement of immobilized LAC and GOx on PANFs 126

The activity was calculated from the time-dependent change of absorbance, and the stabilities 127

of samples were checked by measuring the residual activity time-dependently after incubation in 128

buffer solution at room temperature. The measurement of LAC activity was based on the 129

oxidation of syringaldazine [14]. Syringaldazine (7.8 mg) dissolved in methanol (10 ml) with a 130

final concentration of 0.216 mM. 100 L of the solution containing the immobilized LAC on 131

PANFs (0.1 mg/ml) was mixed with 800 L of 100 mM PB solution (pH 6.5) and the mixtures 132

were heated at 30 C for 10 mins. This heated mixture was added with 100 L of syringaldazine 133

solution (0.216 mM) and the absorbance at 530 nm (A530) was measured by using UV 134

spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). 135

The activity of immobilized GOx on PANFs was measured by GOx assay [15]. The 136

measurement of activity needs a reaction cocktail containing TMB and glucose solution. 137

Reaction cocktail was made of 12 ml of TMB solution (0.576 mg/ml) and 2.5 ml glucose 138

solution (110.1 mg/ml). To measure activity of immobilized GOx, 890 L of reaction cocktail 139

was mixed with 10 L HRP solution (3.798 mg/ml). Then, 100 L of the solution containing the 140

immobilized GOx on PANFs (1 g/ml) was added to 900 L of the mixed solution. The 141

absorbance of immobilized GOx was measured at 655 nm (A655) by using UV spectrophotometer. 142

143

2.5. Preparation of enzyme electrodes 144

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Carbon papers (CPs, thickness of 370 m, 0.44 g/cm3) were treated with acid before use. In 145

a typical preparation, 2cm 2cm squares of CPs was added to an acid solution composed of 146

H2SO4 (98%, 30 ml) and HNO3 (70%, 10 ml) for overnight at room temperature under a stirring 147

condition. Then, acid-treated CPs were washed with distilled water, dried at vacuum condition 148

and stored at room temperature until use. To prepare the GOx-based anode, the immobilized 149

GOx on PANF sample was mixed with Nafion solution (final conc. 0.5 wt %) and this mixture 150

was stored at 4 C for 1 hr. Acid-treated CPs (0.332 cm2) was soaked into the mixture for 10 151

mins, followed by drying at ambient conditions. After drying, the prepared GOx-based enzyme 152

anode was stored in 100 mM PB solution (pH 7.0) at 4 C. For the LAC-based cathode, ABTS 153

(30 mM, 3.3 mg) was added to the mixture of Nafion and immobilized LAC on PANF sample. 154

When the LAC-based cathode containing ABTS was dried, it was stored in 100 mM PB solution 155

(pH 7.0) at 4 C. Since ABTS has high solubility in aqueous solution, washing was not carried 156

out [16]. 157

158

2.6. Biofuel cell operation measurement 159

The electrochemical measurements were performed by using Bio-Logic SP-150 (Knoxville, 160

TN, USA). The performance of enzymatic biofuel cells was measured by circulating 200 mM 161

glucose solution with and without 10 mM BQ in 100 mM PB solution (pH 7.0) at the flow rate of 162

0.4 ml/min within the GOx-based anode. For the LAC-based cathode, the air-breathing structure 163

was used to utilize the ambient air as its oxygen source. The Bio-Logic SP-150 was used to 164

measure the current and voltage outputs of biofuel cell by 3 minute interval under the various 165

load conditions. The power density (W/cm2) was calculated by multiplying current and voltage 166

and then divided by surface of electrode (0.332 cm2). 167

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3. Results and discussion 168

3.1. Immobilization of LAC and GOx on PANFs 169

Figure 1 shows schematic illustrations for the immobilization of enzymes (LAC and GOx) 170

in three different enzyme immobilization methods: EA, EAC, and EAPC. The scanning electron 171

microscope (SEM) images of PANFs, EA, EAC and EAPC were shown in Figure 2 for both 172

GOx and LAC samples. The nanofiber morphology of EA and EAC samples was fairly similar 173

with pristine PANFs (SEM image of pristine PANFs is not shown), whereas EAPC showed 174

remarkably thicker nanofibers revealing the enzyme coating layer over the surface of PANFs. By 175

checking twenty samples of nanofiber images, the average thicknesses of EA-LAC, EAC-LAC 176

and EAPC-LAC were estimated to be 61 6, 82 7 and 115 6 nm, respectively, while those of 177

EA-GOx, EAC-GOx and EAPC-GOx were 75 5, 91 7 and 142 15 nm, respectively. The 178

thickness of the enzyme coating layer for EAPC samples increased significantly than those of EA 179

and EAC samples due to their improved enzyme loading induced by the ammonium sulfate 180

assisted enzyme precipitation step to form the enzyme aggregates and its subsequent chemical 181

crosslinking step. Since the only difference between EAC and EAPC samples was the addition of 182

the enzyme precipitation process for EAPC sample, the SEM data clearly indicates that the 183

enzyme precipitation process is a critical step in order to form the thick enzyme-coating layer 184

over the supporting material. Furthermore, it is interesting to note that EAPC samples with GOx 185

offer a much thicker enzyme coating layer than that of EAPC samples with LAC. GOx can be 186

crosslinked more rigorously than LAC because the number of lysine residues per each GOx 187

molecule is 15 [17], while each LAC molecule has only 8 lysine residues [18]. 188

189

3.2. Activity and stability of immobilized LAC and GOx on PANFs 190

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Activity of immobilized LAC and GOx on PANFs is shown in Figure 3. The activities of 191

EA-LAC, EAC-LAC and EAPC-LAC samples were 1.9, 2.4 and 61.4 A530/min per mg of PANFs, 192

respectively (Fig. 3a). The activity of EAPC-LAC sample was 32 and 25 times higher than that 193

of EA-LAC and EAC-LAC samples, respectively. The activities of immobilized GOx on PANFs 194

have similar tendencies (Fig. 3b). The activities of EA-GOx, EAC-GOx and EAPC-GOx were 195

35.9, 124.6 and 5930 A655/min per mg of PANFs. EAPC-GOx approach offers 165 and 47 times 196

higher activity than that of EA-GOx and EAC-GOx approaches, respectively. The higher activity 197

of EAPC than that of EA and EAC can be interpreted in terms of enhanced enzyme loading by 198

the combination of the precipitation and crosslinking processes. These results matched well with 199

morphological changes of EA, EAC and EAPC samples shown in their corresponding SEM 200

images (Figure 2). 201

Figure 4 shows the stabilities of EA-LAC, EAC-LAC and EAPC-LAC over the 78 days at 202

room temperature. After 78 days, EAPC-LAC maintained 43% of the initial activity, whereas 203

EA-LAC and EAC-LAC maintained 5% and 12% of their initial activities, respectively. The 204

inactivation profiles of all samples were bi-phasic with faster inactivation followed by the slower 205

inactivation at the later phase. The faster inactivation in the early phase can be explained by the 206

labile form of enzymes after being immobilized. The half-life of each sample in the early phase 207

was estimated from the first-order inactivation kinetics. The estimated half-lives of EA-LAC, 208

EAC-LAC and EAPC-LAC were 6, 21 and 53 days, respectively. The stability of immobilized 209

GOx on PANFs was measured in our previous work [12]. After 56 days, the relative activities of 210

EA-GOx, EAC-GOx and EAPC-GOx samples were 22%, 19% and 91%. EA and EAC 211

approaches resulted in poor enzyme stability due to the denaturation and continuous leaching of 212

enzymes from PANFs. Furthermore, the higher enzyme stability offered by EAPC approach can 213

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be explained in terms of its effective enzyme precipitation and crosslinking steps on PANFs. 214

Precipitation by ammonium sulfate allows enzyme molecules to be closely packed. This closely 215

packed and large enzyme aggregates can make the formation of multi-point chemical linkages on 216

the surface of each enzyme molecule more effective when they are treated with glutaric 217

dialdehyde (glutaraldehyde, GA) for the crosslinking of enzymes. These multi-point chemical 218

linkages on the enzyme surface can effectively prevent denaturation and leaching of enzymes, 219

thus stabilizing the activity of enzymes over the long operation time as it was demonstrated by 220

our EAPC samples [19,20]. 221

222

3.3. Enzymatic biofuel cells 223

Figure 5 shows the schematic of enzymatic biofuel cell. Based on the activity and stability 224

tests, both GOx and LAC showed the best performances when they were immobilized in the 225

form of EAPC. Thus, we fabricated the enzyme anode and enzyme cathode by entrapping 226

EAPC-GOx and EAPC-LAC over the carbon paper using Nafion as a binder, respectively. As 227

indicated in Figure 5, the enzymatic biofuel cell is made of the anode chamber, anode current 228

collector, enzyme anode, proton exchange membrane (Nafion 117), enzyme cathode, cathode 229

current collector and cathode holder. During the cell operation, the glucose fuel was 230

electrochemically oxidized by GOx to produce two electrons, two protons and by-product (e.g. 231

gluconolactone) over the enzyme anode. Generated electrons from the anode flow to the cathode 232

through the external load circuit to provide the electrical power. At the cathode, both electrons 233

and protons are combined together to form H2O. 234

Figure 6a and Figure 6b show the voltage-current (V-I) curves and the power density-235

current (P-I) curves of enzymatic biofuel cell with EAPC-GOx and EAPC-LAC at room 236

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temperature, respectively. Without any mediator, the open circuit voltage (OCV) is about 0.34 V 237

and the maximum power density is 3.1 W/cm2. According to previous work [12], we 238

demonstrated that the power density output depends on the enzyme loading. As the enzyme 239

loading increases per unit weight of PANFs, the enzyme activity also proportionally increases 240

per unit weight of PANFs up to a certain value of enzyme loading. For the anode case, the 241

enzyme activity is closely related with electron generation rate and it represents the maximum 242

amount of electrons that can be generated without considering the charge transfer resistance. We 243

speculate that the large amount of electrons generated per unit time by the high enzyme loading 244

of EAPC-based electrode increases the probability of collecting these electrons at the current 245

collector. However, to significantly increase the power density output of our enzymatic biofuel 246

cell, it is also important to improve the electron transfer rate of its electrodes because only a 247

limited amount of electrons would be collected if the enzyme electrodes have poor electron 248

transfer rates regardless of the enzyme activities. 249

In order to increase the electron transfer rates of both enzyme electrodes, the mediators have 250

been utilized [2,21,22]. For the present study, BQ and ABTS were used as the electron transfer 251

mediators for the enzyme anode and enzyme cathode, respectively [23-25]. BQ is a liquid 252

mediator, and it is mixed with the 200 mM of glucose fuel, while ABTS is a solid mediator 253

where it is simultaneously entrapped with EAPC-LAC samples using Nafion binder over the 254

carbon paper surface. The performance of biofuel cells containing EAPC-GOx and EAPC-LAC 255

with mediators is also shown in Figures 6. According to the voltage-current (V-I) curves of 256

enzymatic biofuel cells shown in Figure 6a, when the ABTS is incorporated to the enzyme 257

cathode containing EAPC-LAC, the OCV increases from 0.34 to 0.50 V. However, its current 258

density rapidly drops in a similar manner as the cell without the ABTS-mediated cathode. On the 259

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other hand, when both the BQ and ABTS are incorporated to the enzyme anode and enzyme 260

cathode, respectively, the enzymatic biofuel cell shows a smaller cell voltage drop as the current 261

density increases compared to that of the cell with just ABTS-mediated cathode. This result 262

suggests that the overall cell performance is mainly limited by the slower electron transfer rate of 263

EAPC-GOx-based anode. Due to this smaller overpotential, the enzymatic biofuel cell with both 264

BQ- and ABTS-mediated electrodes generates a much higher current density output as shown in 265

Figure 6a. 266

Figure 6b shows the power density-current (P-I) curves of enzymatic biofuel cells. The 267

power density output of the biofuel cell with just ABTS-mediated cathode does not improve 268

much compared to the biofuel cells without the mediators. Consequently, the maximum power 269

density outputs of the biofuel cells with no mediators and with just ABTS-mediated cathode are 270

3.1 and 5.7 W/cm2, respectively. However, the biofuel cells with BQ- and ABTS-mediated 271

electrodes containing EAPC-GOx and EAPC-LAC showed the maximum power density up to 272

37.4 W/cm2 due to its decreased overpotential mainly offered by the improved electron transfer 273

rate of the anode in the presence of the mediator. 274

275

3.4. Performance stability of enzymatic biofuel cells 276

To confirm the performance stability of enzymatic biofuel cells with EAPC-GOx and 277

EAPC-LAC electrodes, the enzyme electrodes containing EAPC-GOx and EAPC-LAC were 278

incubated in the aqueous buffer under two different temperatures (room temperature and 4 C) 279

over 28 days (Fig. 7). In a 7-day interval, these enzyme electrodes were taken out from the 280

incubation and integrated into our biofuel cell shown in Figure 5 to measure its maximum power 281

density using 200 mM glucose solution at ambient temperature. After the biofuel cell test, both 282

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EAPC-GOx and EAPC-LAC electrodes were removed from the cell and washed with 100 mM 283

PB solution (pH 7.0 for the anode and pH 6.5 for the cathode) to remove any residue glucose 284

solution before they were placed back into the incubation. When incubated over 28 days, the 285

maximum power density output of the biofuel cell with the electrodes stored at room temperature 286

maintained 54% of its initial value, while the biofuel cell with the electrodes stored at 4 C 287

maintained 70% of its initial value. As seen in Figure 7, there is almost no difference in the 288

maximum power density of biofuel cell between two different temperatures used for the thermal 289

stability tests within the 7 days. However, as the thermal stability test continues beyond 7 days, 290

the power density output of the biofuel cell with the electrodes stored at 4 C shows a lower 291

deactivation rate than the biofuel cell with the electrodes stored at room temperature. Overall, the 292

biofuel cell with EAPC-GOx and EAPC-LAC electrodes shows a good performance stability, 293

which agrees with their stability results shown in Figure 3. 294

295

4. Conclusions 296

In this study, we introduced biofuel cells with anode electrode and cathode electrode based 297

on immobilized GOx and LAC on PANFs in the form of enzyme adsorption, precipitation and 298

crosslinking (EAPC). EAPC approach demonstrates that both the loading and stability of GOx 299

and LAC on PANFs can be significantly improved by introducing the enzyme precipitation and 300

crosslinking steps for their immobilization processes. By applying EAPC method to the 301

enzymatic biofuel cell, we have achieved both high power density output and improved 302

performance stability. As shown in successful application to biofuel cells, it is anticipated that 303

EAPC method can be utilized for various types of enzyme-based electrochemical applications 304

such as biosensors and enzyme logic gates. 305

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Acknowledgement 307

This work was supported by the grant from the Agency for Defense Development (ADD -14-70-308

04-01). 309

310

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[19] Mozhaev VV, Melik-nubarov NS, Sergeeva MV, iknis V, Martinek K. Strategy for 349

Stabilizing Enzymes Part One: Increasing Stability of Enzymes via their Multi-Point Interaction 350

with a Support. Biocatal Biotransform 1990;3:179-87. 351

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Surface of a Polymer Support in a Multipoint Fashion. Biochim Biophys Acta 1977;485:1-12. 354

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carbon paper electrodes modified with conducting polymer and its application to a glucose fuel 356

cell. Bioelectrochemistry 2008;74:66-72. 357

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Rev Anal Chem 2012;5:157-79. 359

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their applications: A review. Appl Biochem Microbiol 2007;43:523-35. 361

[24] Lau KT, de Fortescu SAL, Murphy LJ, Slater JM. Disposable glucose sensors for flow 362

injection analysis using substituted 1,4-benzoquinone mediators. Electroanalysis 2003;15:975-81. 363

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oxygen to water. Chem Mater 2007;19:1565-70. 365

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Figure Captions 366

Figure 1. Schematic illustrations for three different enzyme immobilization methods using 367

PANFs: enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC) and enzyme 368

adsorption, precipitation and crosslinking (EAPC). Magnified figure of EAPC represents 369

crosslinking between enzymes molecules. 370

Figure 2. SEM images of (a) EA-LAC, (b) EAC-LAC, (c) EAPC-LAC, (d) EA-GOx, (e) EAC-371

GOx and (f) EAPC-GOx. The scale bars in all images correspond to 1 m. 372

Figure 3. The activities of (a) EA-LAC, EAC-LAC and EAPC-LAC, and (b) EA-GOx, EAC-373

GOx and EAPC-GOx. 374

Figure 4. Stabilities of EA-LAC, EAC-LAC and EAPC-LAC on PANFs at room temperature. 375

Figure 5. Schematic illustrations of biofuel cells consisting of EAPC-GOx (anode electrode) and 376

EAPC-LAC (cathode electrode). 377

Figure 6. (a) The voltage-current (V-I) curves and (b) the power density-current (P-I) curves of 378

enzymatic biofuel cell containing EAPC-GOx and EAPC-LAC with and without mediators (BQ 379

for the anode mediator and ABTS for the cathode mediator). 380

Figure 7. Relative maximum power density of biofuel cells containing EAPC-GOx (anode) and 381

EAPC-LAC (cathode), which were stored at room temperature and 4 C over 28 days. 382

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383

384

Fig. 1. Schematic illustrations for three different enzyme immobilization methods using PANFs: 385

enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC) and enzyme adsorption, 386

precipitation and crosslinking (EAPC). Magnified figure of EAPC represents crosslinking 387

between enzymes molecules. 388

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389

Fig. 2. SEM images of (a) EA-LAC, (b) EAC-LAC, (c) EAPC-LAC, (d) EA-GOx, (e) EAC-390

GOx and (f) EAPC-GOx. The scale bars in all images correspond to 1 m. 391

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392

393

Fig. 3. The activities of (a) EA-LAC, EAC-LAC and EAPC-LAC, and (b) EA-GOx, EAC-GOx 394

and EAPC-GOx. 395

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396

Fig. 4. Stabilities of EA-LAC, EAC-LAC and EAPC-LAC on PANFs at room temperature. 397

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398

Fig. 5. Schematic illustrations of biofuel cells consisting of EAPC-GOx (anode electrode) and 399

EAPC-LAC (cathode electrode). 400

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401

Fig. 6. (a) The voltage-current (V-I) curves and (b) the power density-current (P-I) curves of 402

enzymatic biofuel cell containing EAPC-GOx and EAPC-LAC with and without mediators (BQ 403

for the anode mediator and ABTS for the cathode mediator).. 404

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405

Fig. 7. Relative maximum power density of biofuel cells containing EAPC-GOx (anode) and 406

EAPC-LAC (cathode), which were stored at room temperature and 4 C over 28 days. 407

kim 2014

Documents

enzyme adsorption ea

enzyme anode glucose

accepted date

crosslinking ofglucose

korea university

accepted manuscript1

accepted manuscripttitle

crosslinking eapc approaches