ResearchPap

er

New Biotechnology �Volume 30, Number 5 � June 2013 RESEARCH PAPER

Starchy biomass-powered enzymaticbiofuel cell based on amylases andglucose oxidase multi-immobilizedbioanodeKazuhiro Yamamoto, Takuya Matsumoto, Shota Shimada,Tsutomu Tanaka and Akihiko Kondo

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan

The present study reports the design of a novel bioanode to directly utilize starch as a fuel in an

enzymatic biofuel cell. The enzymatic fuel cell is based on three enzymes (alpha-amylase, glucoamylase

and glucose oxidase). The carbon paste electrode containing these three enzymes and tetrathiafulvalene

can both saccharize and oxidize starchy biomass. In cyclic voltammetry, catalytic currents were

successfully observed with both glucose and starchy white rice used as a substrate. Finally, a membrane-

less white rice/O2 biofuel cell was assembled and the electrochemical performance was evaluated. The

three enzyme based electrode was used as a bioanode and an immobilized bilirubin oxidase (derived

from Myrothecium verrucaria) electrode was used as a biocathode. The biofuel cell delivered an open

circuit voltage of 0.522 V and power density of up to 99.0 mW cm�2. Our results show that a readily

available fuel can be used for enzymatic fuel cells, and will lead to new designs.

IntroductionBiofuel cells are based on bio-functional anodes and cathodes

(bioanodes and biocathodes), which convert organic substrates

to electrical energy. Bioanodes used in microbial fuel cells are

based on microorganisms that can oxidize bioorganic substrates

and generate currents [1,2]. In addition, bioanodes used in enzy-

matic biofuel cells are based on oxidizing enzymes that generate

current by oxidizing substrates such as alcohols or carbohydrates

[3]. Enzymatic biofuel cells have the attractive property of operat-

ing under physiological conditions. By harnessing this property,

enzymatic biofuel cells can be developed for implantable applica-

tions [4]. In addition, the simple mechanism of bioanodes and the

availability of a variety of biological materials for use as substrates

render them as attractive potential energy sources. Basically, enzy-

matic bioanodes are composed of an oxidizing enzyme and a redox

mediator. The diversity of oxidizing enzymes enables the use of a

variety of fuels. Enzymatic glucose fuel cells have been particularly

well-characterized. These bioanodes utilize either glucose oxidase

(GOx) or glucose dehydrogenase (GDH) as the oxidizing enzyme

Corresponding author: Tanaka, T. (tanaka@kitty.kobe-u.ac.jp)

1871-6784/$ - see front matter � 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nbt.2013.0

[5,6], while alcohol dehydrogenase is utilized in alcohol fuel cells

and fructose dehydrogenase is utilized in fructose fuel cells [7,8].

Although several enzymatic monosaccharide (glucose, fructose,

and so on.) and alcohol fuel cells have been described in the

literature, there has been comparatively little study of bioanodes

that can directly oxidize oligosaccharides as fuel sources [9,10].

Several works have shown microbial fuel cells operating with

starchy or cellulosic biomass [11,12], but as of yet, only one

enzymatic fuel cells as we understand it. This is because there is

no enzyme that can directly oxidize long-chain polysaccharides.

Starchy or cellulosic biomass is composed of polysaccharides,

and thus represents an environmentally friendly fuel for biocon-

version processes involving enzymes or microorganisms. Starch is

contained in many staple foods (e.g. potatoes, wheat, corn, rice)

and consists of a large number of glucose units conjugated with

glycosidic bonds. However, while most oxidizing enzymes can

utilize monosaccharides, they cannot directly process polysac-

charides. Hence, a saccharification process is required to hydrolyze

polysaccharides into monosaccharides before use as a biofuel. In

the presence of alpha-amylase, starch is hydrolyzed to oligosac-

charides or dextrin, both of which are hydrolyzed to glucose by

4.005 www.elsevier.com/locate/nbt 531

RESEARCH PAPER New Biotechnology � Volume 30, Number 5 � June 2013

Research

Pap

er

glucoamylase. Similarly, in the degradation of cellulosic materials

into glucose, several kinds of cellulase are required. Recently,

carboxymethyl cellulose (CMC), which is one of the water-soluble

cellulosic materials, was utilized as a fuel for enzymatic fuel cells.

However, the bioanode was not able to oxidize CMC, and it

required a multi-step saccharification process using three types

of cellulase to generate electricity from CMC [13]. To simplify this

multi-step process, which requires saccharification of polysacchar-

ides and utilization of monosaccharides, biochemical engineers

have developed bioconversion processes in which polysaccharides

are simultaneously saccharized and fermented (SSF). Bioconver-

sion processes have been developed involving recombinant micro-

organisms in which alpha-amylase and glucoamylase genes can be

induced to enable direct utilization of starchy biomass. These

processes have been used to successfully convert starchy biomass

into bio-ethanol fuel [14,15]. In this study, a novel bioanode

inspired by the above-mentioned SSF process was designed to

directly utilize starch as a fuel for an enzymatic fuel cell. The

novel bioanode enables simultaneous saccharification and oxidi-

zation of starch on the electrode surface. Here, we demonstrate

that the enzymatic fuel cell can operate using a suspension of

white rice as fuel, with a multi-immobilized bioanode of hydro-

lases and oxidase. Using multi-immobilization of GOx, alpha-

amylase and glucoamylase on a carbon paste electrode (CPE),

we developed an electrode that can both saccharize and oxidize

starchy biomass.

Materials and methodsConstruction of expression plasmidsKOD FX polymerase (TOYOBO Co., Ltd., Osaka, Japan) was used

for PCR, and the PCR-amplified sequence was verified by DNA

sequencing. The expression plasmid for Alpha-amylase (AmyA)

derived from Streptococcus bovis 148 was constructed as follows.

The gene encoding AmyA was obtained by PCR using a vector from

a previous report [14] as a template with the 50 primer (50-GGG

GTA CCG GAT CCG ATA TCG ATG AAC AAG TGT C-30) and the 30

primer (50-CCC AAG CTT GAA TTC TTA TTT TAG CCC ATC TTT

ATT AT-30). The amplified fragment was subcloned into the KpnI/

EcoRI sites of the pET32b(+) vector (Novagen, San Diego, CA, USA)

to yield pET32b(+)-AmyA.

Expression and purification of enzymesAmyA was expressed and purified as follows. The pET32b(+)-AmyA

plasmid was introduced into Escherichia coli BL21 (DE3). Cells were

grown in LB medium to an OD (600 nm) of 0.5 at 378C, then cells

were incubated an additional 30 min at 258C. Expression of the

protein was induced by the addition of isopropyl b-D-1-thiogalac-

topyranoside (IPTG) to a final concentration of 0.5 mM. After

growth for an additional 24 hours at 258C, cells were harvested

by centrifugation. The cell pellets were resuspended in 50 mM

phosphate, 150 mM NaCl, pH 8.0, and lysed by sonication. AmyA

was purified from the soluble fraction using TALON metal affinity

resin (Takara Bio, Inc., Shiga, Japan) according to the manufac-

turer’s protocol, and dialyzed against 20 mM phosphate, 150 mM

NaCl, pH 8.0. The concentration of purified AmyA was determined

using a BCA protein assay kit (Pierce). AmyA hydrolysis activity

was assayed by an alpha-amylase assay kit (Kikkoman Corp.,

Chiba, Japan).

532 www.elsevier.com/locate/nbt

Bioanode preparationThe CPE containing tetrathiafulvalene (TTF) and GOx from Asper-

gillus niger (GOx, purchased from Nakarai tesuque, Kyoto, Japan),

alpha-amylase from S. bovis 148 (AmyA), and glucoamylase from

Rhizopus oryzae (GluR, TOYOBO Co., Ltd., Osaka, Japan) (GOx/

AmyA/GluR/CPE) was prepared as follows: Enzymes solutions

(containing 75.8 mg GOx, 245 U mg�1; 18.2 mg GluR, 37 U mg�1;

1; or 15 mg AmyA, 70 000 U mg�1) and a solution of TTF (30 mL in a

saturated solution of methanol) were mixed with carbon paste

(BAS Inc., Tokyo, Japan) (60 mg). The carbon paste mixture was

packed into the electrode (diameter 3 mm) and compressed with

clean copy paper. Then the electrode surface was coated with an

aqueous solution of polyarylamine (PAA) (1.0% (w/v) in distilled

water, 10 mL) and glutaraldehyde (GA) (0.5% (w/v) in distilled

water, 1 mL) complex film. Electrodes were allowed to dry for

2 hours at ambient temperature. The CPE containing TTF and

GOx (GOx/CPE) was prepared as follows: Enzymes solution (con-

taining 75.8 mg GOx) and a solution of TTF (30 mL in a saturated

solution of methanol) were mixed with carbon paste (60 mg). This

carbon paste mixture was also packed into the electrode and

compressed with clean copy paper, and then the surface was

coated with PAA and GA complex film.

Biocathode preparationEnzyme solution (containing 45 mg billirubin oxidase (BOx) from

Myrothecium verrucaria, 5.6 U mg�1, Sigma–Aldrich Corp., St.

Louis, MO, USA) and a solution of 2,20-azino-bis(3-ethylbenzotha-

zolin-6-sulfonic acid) (ABTS) (30 mL in a saturated solution of

water) containing carbon paste (60 mg) were packed into the

electrode and compressed with clean copy paper. Then the elec-

trode surface was coated with an aqueous solution of PAA (1.0%

(w/v) in distilled water, 10 mL) and GA (0.5% (w/v) in distilled

water, 1 mL) complex film. Electrodes were allowed to dry for

2 hours at ambient temperature.

Electrochemical measurementsThe electrochemical measurements were performed using a

Model 2323 Bi-Potentiostat (BAS Inc.) and a conventional

three-electrode cell. The Ag/AgCl electrode and a Pt wire were

used as the reference electrode and the counter electrode, respec-

tively. The bioanode described above was used as the substrate of

the working electrode. Cyclic voltammogram (CV) analysis was

used to characterize the electrocatalytic properties of the bioa-

nodes with respect to glucose or starchy biomass oxidation, and

CV curves ranging between �0.2 V and +0.35 V were measured.

Open circuit potential analysis was used to measure the electro-

motive force of cells consisting of a bioanode and a biocathode,

and measurement time was 10 s. Linear sweep voltammogram

analysis was done to measure the maximum power density of

cells. All electrochemical measurements were conducted at ambi-

ent temperature and physiological pH.

Preparation of rice crude solutionPolished white rice (kindly provided from Hyogo Prefecture,

Japan) was ground to produce flour with a particle size of approxi-

mately 0.5 mm using a laboratory disintegrator. The flour was

suspended in phosphate buffer and electrochemical determina-

tion was performed.

New Biotechnology �Volume 30, Number 5 � June 2013 RESEARCH PAPER

FIGURE 1

Direct production of electrical current from starchy biomass using a GOx/

AmyA/GluR-multi-immobilized bioanode. ResearchPap

er

Results and discussionPreparation of anodic enzymesAmyA derived from S. bovis 148 was utilized, which can efficiently

adsorb and hydrolyze starch [16,17]. AmyA was produced by

recombinant E. coli, and purified AmyA retained efficient hydro-

lysis activity (70 000 U mg�1). In addition, glucoamylase from R.

oryzae (GluR) was utilized, which is a very efficient hydrolytic

enzyme. Starchy biomass has been efficiently hydrolyzed to glu-

cose by a combination of AmyA and GluR in previous studies

[14,15]. The GOx that we utilized is produced by A. niger. GOx is a

flavin adenine dinucleotide (FAD)-binding enzyme, and has been

used as a glucose sensor and in enzymatic glucose fuel cells [18,19].

The design of three enzyme co-immobilized bioanodeThe design of the bioanode is illustrated in Fig. 1. Production of

the bioanode required the multi-immobilization of the three

enzymes (AmyA, GluR and GOx) in a densely packed configura-

tion while retaining the enzymatic activity. TTF was also immo-

bilized on the bioanode to serve as a redox mediator, because

oxidized derivatives of TTF had high electrical conductivity.

Glassy carbon electrode is often utilized for the preparation of

biosensor or bioelectrode of biofuel cells, however, obtaining a

sufficient density of enzymes on a surface such as glassy carbon is

usually difficult because of surface area constraints. CPEs repre-

sent an attractive alternative for multi-immobilization of

FIGURE 2

Cyclic voltammograms of the GOx/AmyA/GluR/CPE (a) or GOx/CPE (b) in 0.1 M, pH

starch (weight equivalent to 50 mM glucose: red line). (c) Cyclic voltammograms of tbuffer. Data were collected at 10 mV s�1.

enzymes because multiple enzymes can be densely packed by

kneading them with a carbon powder [20–22]. Paraffin modified

simple carbon powder was utilized in this study, although a

porous carbon powder is more suitable to expand the surface area

of the electrode. To reduce the potential for deactivation of

enzymes by adsorption to the carbon paste, enzymes were

assembled using GA crosslinking of their amino groups, which

increased enzymatic stability. The use of GA crosslinking for the

7.2, phosphate buffer (blue line) containing 50 mM glucose (green line) or raw

he BOx/CPE in N2 (blue line) or O2 (red line) saturated 0.1 M, pH 7.2, phosphate

www.elsevier.com/locate/nbt 533

RESEARCH PAPER New Biotechnology � Volume 30, Number 5 � June 2013

FIGURE 3

The assembly of a membrane-less white rice/O2 biofuel cell.

FIGURE 4

The power density curve of the white rice/O2 biofuel cell obtained by LSV in

0.1 M, pH 7.2, phosphate buffer containing 50 mM white rice (weight

equivalent to 50 mM glucose). Data were collected at 10 mV s�1.

Research

Pap

er

assembly and stabilization of enzymes has been reported else-

where [23,24]. Another significant concern when using CPE is the

potential for exsorption of the enzymes and/or mediator from the

electrode surface. To prevent exsorption, CPE can be covered with

a thin membrane such as a nitrocellulose film or a poly-ion

complex (PIC) membrane [25]. In this study, we covered the

CPE with a PAA membrane, which can be easily produced by

gelatification of PAA with the addition of GA.

Cyclic voltammetry of multi-functional bioanodeThe electrochemical performance of the bioanode was assessed

using CV analysis. The analysis was conducted in a 0.1 M phosphate

buffer (pH 7.2). GOx/AmyA/GluR/CPE was tested for direct oxida-

tion of raw starchy biomass. Hyogo Kinuhikari white rice was

chosen as a model of starchy biomass. The results of CV analysis

of simultaneous saccharification and oxidation of white rice and

glucose using the GOx/AmyA/GluR/CPE are shown in Fig. 2a. Cat-

alytic currents were observed with both glucose and white rice used

as a substrate. However, with a GOx-only immobilized bioanode,

GOx/CPE, no catalytic current was observed in the presence of white

rice (Fig. 2b). These results indicated that simultaneous saccharifica-

tion and oxidation of white rice requires co-immobilization of GOx

along with AmyA and GluR. Although anodic currents were

observed in the presence of white rice with the GOx/AmyA/

GluR/CPE, this current was lower than that obtained with glucose,

although equivalent initial amounts of glucose and raw starch were

used. Polished white rice is also composed of water, protein, lipid

and ash. The usable raw starch component is 80%, and therefore we

expected that the anodic current obtained with white rice would be

80% of that obtained with glucose. Actually, the obtained anodic

current from white rice was approximately 70% of that obtained

with glucose. Thus, GOx/AmyA/GluR/CPE efficiently operated even

with white rice suspended in crude solution, showing that a solid–

solute reaction (degradation of starchy biomass to glucose) and a

solute–solute reaction (oxidation of glucose) occurred consecu-

tively. Incidentally, the extra addition of AmyA or GluR in the

bioanode solution did not result in more efficient simultaneous

saccharification and oxidation of white rice (data not shown). This

result indicated that the addition of oxidase and hydrolase con-

tained in the bioanode was suitable for simultaneous saccharifica-

tion and oxidation. Even more remarkable, with GOx/CPE in

addition to AmyA and GluR in the solution, a poor catalytic current

was observed in the presence of white rice (data not shown). This

result clearly indicated that multi-immobilization of GOx, AmyA

and GluR was an effective approach for simultaneous saccharifica-

tion and oxidation of starchy biomass.

The assembly of a membrane-less white rice/O2 biofuel cellTo evaluate the electrochemical performance as an enzymatic bio-

fuel cell operating with raw starch biomass, linear sweep voltam-

metry (LSV) was used. GOx/AmyA/GluR/CPE was used for a

bioanode, and bilirubin oxidase derived from M. verrucaria (BOx)

and ABTS immobilized CPE (BOx/CPE) was used for a biocathode

[26,27]. ABTS is commonly used as a substrate for peroxidase, laccase

and bilirubin oxidase, and serves as an electron donor. The BOx/CPE

performance was assessed by CV in a 0.1 M phosphate buffer (pH

7.2). The result of CV analysis of O2 oxidation is shown in Fig. 2c.

Catalytic current was observed only in the O2 saturated condition,

534 www.elsevier.com/locate/nbt

indicating that the BOx/CPE was suitable as a biocathode of our

biofuel cell. To assemble a membrane-less white rice/O2 biofuel cell,

these two electrodes were immersed in O2 saturated 0.1 M phosphate

buffer (pH 7.2) containing 50 mM white rice (weight equivalent to

50 mM glucose) (shown in Fig. 3). Fig. 4 shows the current and the

power density of the biofuel cell as a function of the operating cell

voltage (I–V and P–V curve) at room temperature. As shown in Fig. 4,

open-circuit voltage (Voc) and the short-circuit current of the biofuel

cell are 0.522 V and 0.469 mA cm�2, respectively. The maximum

power density of the biofuel cell was 99.0 mW cm�2 at 0.211 V.

Although our designed bioanode saccharized and oxidized starch

biomass simultaneously on the electrode surface, the value of the

power density was almost the same order as those reported in the

literature such as for a gold nanoparticle modified Au electrode,

which can operate without redox mediator [28], and one in which

maltodextrin was used as a power source [10].

In summary, we developed a novel enzymatic biofuel cell that

operates with starchy biomass. The biofuel cell was based on an

AmyA, GluR and GOx multi-immobilized bioanode that can

New Biotechnology �Volume 30, Number 5 � June 2013 RESEARCH PAPER

directly utilize starch. The biofuel cell delivered an open circuit

voltage of 0.522 V and power density of up to 99.0 mW cm�2. Our

results which show that a readily available fuel can be used for

enzymatic fuel cells will promote their study as logic gate systems

such as typical Boolean logic operations which operate with

concerted catalysis of multi-enzyme reaction [29–31] and will

lead to new designs.

AcknowledgementsThis work was supported in part by a Grant-in-Aid for Japan Society

for the Promotion of Science (JSPS) Fellows, by Grant-in-Aid for

Scientific Research(B) and by Special Coordination Funds for

Promoting Science and Technology, Creation of Innovation

Centers for Advanced Interdisciplinary Research Areas (Innovative

Bioproduction Kobe), MEXT, Japan.

References

ResearchPap

er

[1] Logan BE, Hamelers B, Rozendal R, Schroder U, Keller J, Freguia S, et al. Microbialfuel cells: methodology and technology. Environmental Science and Technol-ogy 2006;40:5181–92.

[2] Chaudhuri SK, Lovley DR. Electricity generation by direct oxidation of glucosein mediatorless microbial fuel cells. Nature Biotechnology 2003;21:1229–32.

[3] Willner I, Yan Y-M, Willner B, Tel-Vered R. Integrated enzyme-based biofuelcells – a review. Fuel Cells 2009;9:7–24.

[4] Barton SC, Gallaway J, Atanassov P. Enzymatic biofuel cells for implantable andmicroscale devices. Chemical Reviews 2004;104:4867–86.

[5] Chen C, Wang L, Tan Y, Qin C, Xie F, Fu Y, et al. High-performance ampero-metric biosensors and biofuel cell based on chitosan-strengthened cast thinfilms of chemically synthesized catecholamine polymers with glucose oxidaseeffectively entrapped. Biosensors and Bioelectronics 2011;26:2311–6.

[6] Tan Y, Deng W, Ge B, Xie Q, Huang J, Yao S, et al. Biofuel cell and phenolicbiosensor based on acid-resistant laccase-glutaraldehyde functionalized chito-san-multiwalled carbon nanotubes nanocomposite film. Biosensors and Bioe-lectronics 2009;24:2225–31.

[7] Deng L, Shang L, Wen D, Zhai J, Dong S. A membraneless biofuel cell powered byethanol and alcoholic beverage. Biosensors and Bioelectronics 2010;26:70–3.

[8] Wu X, Zhao F, Varcoe JR, Thumser AE, Avignone-Rossa C, Slade RC, et al. A one-compartment fructose/air biological fuel cell based on direct electron transfer.Biosensors and Bioelectronics 2009;25:326–31.

[9] Rasmussen M, Ritzmann RE, Lee I, Pollack AJ, Scherson D. An implantable biofuelcell for a live insect. Journal of the American Chemical Society 2012;134:1458–60.

[10] Zhu Z, Wang Y, Minteer SD, Zhang YP. Maltodextrin-powered enzymatic fuelcell through a non-natural enzymatic pathway. Journal of Power Sources 2011;196:7505–9.

[11] Ren Z, Ward TE, Regan JM. Electricity production from cellulose in a microbialfuel cell using a defined binary culture. Environmental Science and Technology2007;41:4781–6.

[12] Niessen J, Schroder U, Scholz F. Exploiting complex carbohydrates for microbialelectricity generation – a bacterial fuel cell operating on starch. ElectrochemistryCommunications 2004;6:955–8.

[13] Cheng H, Qian Q, Wang X, Yu P, Mao L. Electricity generation from carbox-ymethyl cellulose biomass: a new application of enzymatic biofuel cells. Elec-tochimica Acta 2012;82:203–7.

[14] Yamada R, Bito Y, Adachi T, Tanaka T, Oginoa C, Fukuda H, et al. Efficientproduction of ethanol from raw starch by a mated diploid Saccharomycescerevisiae with integrated a-amylase and glucoamylase genes. Enzyme andMicrobial Technology 2009;44:344–9.

[15] Yamada R, Tanaka T, Ogino C, Fukuda H, Kondo A. Novel strategy for yeastconstruction using delta-integration and cell fusion to efficiently produceethanol from raw starch. Applied Microbiology and Biotechnology 2010;85:1491–8.

[16] Satoh E, Niimura Y, Uchimura T, Kozaki M, Komagata K. Molecular cloning andexpression of two alpha-amylase genes from Streptococcus bovis 148 in Escherichiacoli. Applied and Environment Microbiology 1993;59:3669–73.

[17] Lin SC, Liu WT, Liu SH, Chou WI, Hsiung BK, Lin IP, et al. Role of the linkerregion in the expression of Rhizopus oryzae glucoamylase. BMC Biochemistry2007;8:9.

[18] Mano N, Mao F, Shin W, Chen T, Heller A. A miniature biofuel cell operating at0.78 V. Chemical Communications 2003;518–9.

[19] Wong CM, Wong KH, Chen XD. Glucose oxidase: natural occurrence, function,properties and industrial applications. Applied Microbiology and Biotechnology2008;78:927–38.

[20] Yang X, Tian G, Jiang N, Su B. Immobilization technology: a sustainablesolution for biofuel cell design. Energy & Environmental Science 2012;5:5540–63.

[21] Liu S, Ju H. Reagentless glucose biosensor based on direct electron transfer ofglucose oxidase immobilized on colloidal gold modified carbon paste electrode.Biosensors and Bioelectronics 2003;19:177–83.

[22] Wang J, Musameh M, Mo JW. Acid stability of carbon paste enzyme electrodes.Analytical Chemistry 2006;78:7044–7.

[23] Kowal R, Parsons RG. Stabilization of proteins immobilized on Sepharose fromleakage by glutaraldehyde crosslinking. Analytical Biochemistry 1980;102:72–6.

[24] Yu CM, Yen MJ, Chen LC. A bioanode based on MWCNT/protein-assisted co-immobilization of glucose oxidase and 2,5-dihydroxybenzaldehyde for glucosefuel cells. Biosensors and Bioelectronics 2010;25:2515–21.

[25] Komaba S, Mitsuhashi T, Shiraishi S. Optimization of enzyme anode andcathode with polyion complex for the application to biofuel cells. Electochem-istry 2008;76:619–24.

[26] Ivnitski D, Artyushkova K, Atanassov P. Surface characterization and directelectrochemistry of redox copper centers of bilirubin oxidase from fungi Myr-othecium verrucaria. Bioelectrochemistry 2008;74:101–10.

[27] Szczupak A, Kol-Kalman D, Alfonta L. A hybrid biocathode: surface display ofO2-reducing enzymes for microbial fuel cell applications. Chemical Commu-nications 2012;48:49–51.

[28] Wang X, Falk M, Ortiz R, Matsumura H, Bobacka J, Ludwig R, et al. Mediatorlesssugar/oxygen enzymatic fuel cells based on gold nanoparticle-modified electro-des. Biosensors and Bioelectronics 2012;31:219–25.

[29] Zhou M, Zheng X, Wang J, Dong S. A self-powered and reusable biocomputingsecurity keypad lock system based on biofuel cells. Chemistry A EuropeanJournal 2010;16:7719–24.

[30] Halamek J, Tam TK, Strack G, Bocharova V, Pita M, Katz E, et al. Self-poweredbiomolecular keypad lock security system based on a biofuel cell. ChemicalCommunications 2010;46:2405–7.

[31] Strack G, Pita M, Ornatska M, Katz E. Boolean logic gates that use enzymes asinput signals. Chembiochem 2008;9:1260–6.

www.elsevier.com/locate/nbt 535