Journal of Electrochemistry
Volume 17
Issue 3 Special Issue of Chemo/Biosensing
Technology(Editor: Professor ZHANG Zongrang)
2011-08-28
Development of dehydrogenase-based bioanode using
poly(phenosafranin)-functionalized SWCNT nanocomposites and
its application to ethanol biosensor
S. Saleh Farhana
Okajima Takeyoshi
Mao Lanqun
Takeo Ohsaka
Recommended Citation
S. Saleh Farhana, Okajima Takeyoshi, Mao Lanqun, and Takeo Ohsaka (2011) "Development of
dehydrogenase-based bioanode using poly(phenosafranin)-functionalized SWCNT nanocomposites and
its application to ethanol biosensor," Journal of Electrochemistry: Vol. 17: Iss. 3, Article 2.
Available at: https://jelectrochem.xmu.edu.cn/journal/vol17/iss3/2
This Special Issue Article is brought to you for free and open access by Journal of Electrochemistry. It has been
accepted for inclusion in Journal of Electrochemistry by an authorized editor of Journal of Electrochemistry.
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2011 $ 8 %
ELECTROCHEMISTRY
No. 3
Aug. 2011
Artical ID:10063471(2011)03026308
Development of DehydrogenaseBased Bioanode Using Poly
( Phenosafranin) Functionalized SWCNT Nanocomposites
and its Application to Ethanol Biosensor
Farhana S. Saleh1 , Takeyoshi Okajima1 , MAO Lanqun2 , Takeo Ohsaka1
(1. Department of Electronic Chemistry, Interdisciplinary Graduate School of Science and Engineering,
Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 2268502, Japan;
2. Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, the Chinese Academy of
Sciences( CAS) , Beijing 100190, China)
Abstract:
A New type of dehydrogenasebased amperometric ethanol biosensor was constructed u
sing alcohol dehydrogenase ( ADH) which was immobilized on the edgeplane pyrolytic graphite ( EP
PG) electrode modified with poly( phenosafranin) functionalized singlewalled carbon nanotube ( PPS
SWCNT) . The PPSSWCNT modified EPPG electrode was prepared by electropolymerization of phe
nosafranin on the EPPG electrode which was previously coated with SWCNT. The performance of the
ADH / PPSSWCNT / EPPG electrode was evaluated using cyclic voltammetry and amperometry in the
presence of ethanol. The fabricated ethanol biosensor provided a reasonable sensitivity of 2. 0 μA·
cm - 2 ·mmol - 1 ·L and a low detection limit (36 μmol·L - 1 ) for the electrocatalytic oxidation of eth
anol with a linear concentration dependence upto ~ 1. 0 mmol·L - 1 at a detection potential of 0. 2 V.
Key words: phenosafranin; electropolymerization; NADH; SWCNT; electrocatalysis
CLC Number: O646; TP212. 3
Document Code: A
1
Introduction
The quantitative measurement of alcohol is very
important for clinical and forensic purposes in the an
alyses of human breath [1] . Ethanol is an important
compound in medicine, biotechnology and the food
industry, etc. ,and it is often monitored for toxicologi
cal and psychological effects [2] . In certain industrial
fields, such as fermentation and distillation, the etha
ven alcohol poisoning [3] . Some analytical methods
have been developed during the years for the determi
nation of ethanol. An amperometric enzymebased
biosensor is one of the best choices for biochemical a
nalysis due to their good selectivity, sensitivity, rapid
response, miniature size, and reproducible results [4] .
The good selectivity is attributed to the specific catal
ysis by the enzyme.
nol concentration can reach toxic levels, causing in
In most cases, NAD + ( the oxidized form of β
nicotinamide adenine dinucleotide ( NADH ) ) de
junctiva, irritation of the skin and, at high levels, e
reagent for their functioning. Electrode modification
flammation of the nasal mucous membrane and con
Received: 20100607
pendent enzymebased biosensors require NADH as a
Corresponding author, Tel:(8145)9245404, Email:ohsaka@ echem. titech. ac. jp
The present work was financially supported by GrantinAid for Scientific Research ( A) ( No. 19206079) from the Ministry of Edu
cation, Culture, Sports, Science and Technology ( MEXT) , Japan, Tokyo Institute of Technology Global COE Program for Energy
Science, and JapanChina Research Program on Enzymebased Biofuel Cells organized and sponsored by Japan Science and Tech
nology ( JST) and Natural Science Foundation of China ( NSFC) .
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with carbon nanotubes ( CNTs ) has been shown to
sensors [2528] .
tion, thus allowing the preferable construction of de
genasebased ethanol biosensor by immobilizing alco
sors
( phenosafranin) ( PPS) functionalized SWCNT nano
produce an electrocatalysis for NADH electrooxida
hydrogenase enzyme based electrochemical biosen
[57]
. Concerning enzyme biosensors based on
The present work aims at developing dehydro
hol dehydrogenase ( ADH) on the surface of the poly
CNTscomposite electrodes, they have been found to
composite. Previously we have reported the electro
quence of the unique properties of CNTs with the
lytic graphite ( BPPG) modified with PPS film pre
possess improved analytical performance as a conse
catalytic oxidation of NADH by the basalplane pyro
wellknown advantages of composite electrode designs
pared by electropolymerization of phenosafranin ( PS)
rent [8] . Many recent studies have aimed at the func
potential of - 0. 458 V at pH 7. 0 [29] . We have also
such as simple renewability and low background cur
which is an Nsubstituted phenazine with a halfwave
tionalization of CNTs with various molecules using co
reported an excellent electrocatalysis of PPSfunction
ches [1112] in order to obtain desired properties. The
( EPPG) electrode for the NADH oxidation [28] . Here,
valent [910] or noncovalent bond formation approa
alized SWCNT modified edgeplane pyrolytic graphite
covalent functionalization is based on the formation of
by immobilizing ADH onto the PPSSWCNT nanocom
cules, while the noncovalent functionalization is
PPSSWCNT / EPPG) biosensor was fabricated. The
chemical bonds between CNTs and functional mole
based on adsorption, wrapping, πstacking, or hy
posite film, dehydrogenasebased ethanol ( ADH /
obtained results showed that the ADH and PPSSWC
drophobic / ππ interactions, etc. Among the many
NT nanocompositebased bioanode possesses an effi
chemical functionalization can provide an efficient,
2
different strategies to functionalize CNTs, electro
clean, and more versatile alternative
[13]
. Fabrication
of CNTs / conducting polymer composites has become
of great interest since the CNTs can improve the elec
trical and mechanical properties of polymers. It has
been demonstrated that the CNTs / conducting polymer
composites possess the original properties of the indi
cient electrocatalytic activity for ethanol oxidation.
2. 1
Experimental
Chemicals and Reagents
Singlewalled carbon nanotube ( SWCNT) with a
diameter = 1. 2 ~ 1. 5 nm and a length = 2 ~ 5
μm were purchased from Aldrich ( Tokyo, Japan) .
Phenosafranin ( PS) was purchased from Acros Or
vidual components with a synergistic function [1416] .
ganics ( Geel, Belgium) . Oxidized form of βnicotin
amide adenine dinuleotide ( NAD + , Oriental Yeast
NTs) / poly (3octylthiophene) composites have been
cohol dehydrogenase ( ADH) from Baker′s yeast, de
For example, singlewalled carbon nanotubes ( SWC
shown to have good photovoltaic properties [17] , and
Co. Ltd. , Tokyo, Japan) was used as received. Al
hydrated ethanol and 25% glutaraldehyde ( GA) so
CNTs / polyaniline composites have been found to be a
lution in water were purchased from Wako ( Japan) .
charge capacity, lowcharge voltage and highdis
Sigma ( USA) . TrisHCl buffer solution ( TBS; 0. 05
good material in batteries with a highchargedis
charge voltage
[18]
. So far, several conducting poly
mers such as polypyrrole, polyaniline, polyphenothia
zine, polythiophene and their derivatives have been
used to fabricate CNTs / polymer composites
[1923]
.
However, there are limited studies on the CNTs / poly
( phenazine ) dye composites
[24]
, although poly
( phenazine) dyes are widely used as redox mediators
in the construction of electrochemical sensors and bio
Bovine serum albumin ( BSA ) was purchased from
mol·L - 1 , pH 8. 2) was used as the supporting elec
trolyte for electrochemical experiments. The solutions
throughout this work were always prepared using dei
onized water from a MilliQ water system ( Millipore,
Japan ) .
Edgeplane pyrolytic graphite ( EPPG )
( Bioanalytical Systems Inc. ( BAS) ; 3 mm in diame
ter) disk was used as the working electrode. All of
the other chemicals were of analytical grade and were
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Farhana S. Saleh &:'()*+,-./0123456789:;<=>?@A
used without further purification.
2. 2
Apparatus and Electrochemical
Measurements
Cyclic voltammetric and amperometric measure
ments were carried out with an ALS CHI 750Cz elec
trochemical analyzer ( Eco Chemie, Ultecht, The
Netherlands) using a conventional twocompartment
threeelectrode system with the prepared bioanodes as
working electrodes, a spiral Pt wire as counter elec
trode and a Ag | AgCl | KCl( sat. ) as reference elec
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trices. For attaching the enzyme layer, typically 4. 5
μL of ADHBSAGA mixture ( 2 μL of 20 mg / mL
ADH solution in 0. 05 mol · L - 1 TBS, pH 8. 2 + 1
-1
μL 1% ( by mass) BSA solution in 0. 05 mol · L
TBS, pH 8. 2 + 1. 5 μL 1% ( by mass) GA ( in wa
ter) was put on the PPSSWCNT / EPPG electrode and
airdried at room temperature for at least 1 h. The e
lectrode assembly fabricated ( named as ADH / PPS
SWCNT / EPPG electrode) is schematically shown in
Scheme 1.
trode. The amperometric measurements of the ethanol
sensor fabricated were performed in 0. 1 mol · L - 1
PBS, stirred at ca. 300 r / min using a magnetic stir
rer. A personal computer was used for data storage
and processing. Electrolyte solutions were deaerated
by bubbling Ar gas ( 99. 99% ) for at least 30 min
prior to electrochemical measurements. All the meas
urements were carried out at room temperature (25 ±
1 ℃ ).
2. 3
Electrode Preparation and Enzyme
Immobilization
The EPPG electrodes were first carefully pol
ished with emery paper and then transferred to 0. 1
mol·L - 1 H2 SO4 solution and the potential scan was
repeated between - 0. 2 and 1. 4 V at 100 mV·s - 1
Scheme 1
3
3. 1
Scheme of the ADHbased composite electrode
for the electrocatalytic oxidation of ethanol
Results and Discussion
Electrocatalytic Characterization of
the ADH / PPSSWCNT / EPPG Elec
trode for Ethanol Oxidation
The response of the ADH / PPSSWCNT / EPPG
until a stable cyclic voltammetric response was at
biosensor was electrochemically tested by cyclic volta
DMF to form a 1 mg / mL SWCNT suspension solution
10 mmol · L - 1 NAD + in the absence (----) and
ified EPPG ( SWCNT / EPPG) electrode was prepared
No electrocatalytic response was observed at the
tained. 1 mg of the SWCNT was dispersed in 1 mL
with the aid of ultrasonic agitation. The SWCNT mod
by casting 30 μL of the SWCNT suspension on the
EPPG electrode surface and airdrying the casted sus
pension solution. A film of PPS was prepared by im
mmetry in 0. 05 mol·L - 1 TBS ( pH 8. 2) containing
presence ( ———) of 20 mmol·L - 1 ethanol ( Fig. 1) .
ADH / PPSSWCNT / EPPG electrode in 0. 05 mol ·
L - 1 TBS ( pH 8. 2) containing 10 mmol·L - 1 NAD +
at a scan rate of a 5 mV·s - 1 in the potential range of
mersing the SWCNT / EPPG electrode in 0. 2 mol ·
interest. Upon addition of 20 mmol·L - 1 of ethanol,
10 min and then by repeating the potential scan be
a large anodic peak with a decrease in the reduction
L - 1 H2 SO4 solution containing 0. 5 mmol·L - 1 PS for
tween - 0. 5 and 1. 3 V at 50 mV · s . The thus
-1
fabricated PPSSWCNT modified EPPG electrode is
the cyclic voltammetric response was characterized by
current. The onset potential of ca. - 0. 2 V vs. Ag |
AgCl | KCl( sat. ) shows a reasonable relevance to that
hereinafter abbreviated as PPSSWCNT / EPPG elec
obtained for NADH oxidation at the PPSSWCNT
immobilized by a crosslinking reaction using glutaral
NADH oxidation is shown in the inset of Fig. 1. Upon
trode. Finally, alcohol dehydrogenase ( ADH ) was
dehyde ( GA) in bovine serum albumin ( BSA) ma
nanocomposite modified electrode.
The result of
the addition of NADH, a dramatic enhancement in
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the anodic peak current is observed which comes from
the mediated oxidation of NADH to NAD + . These re
sults confirmed the efficient electrocatalytic property
of the biocomposite film electrode which is associated
with the oxidation of ethanol via ADH catalysis. ADH
catalyzes, in the presence of NAD + as a coenzyme,
the oxidation of ethanol to acetaldehyde and simulta
neously, NAD + is reduced to NADH. Thus, the a
nodic peak ( solid line) in Fig. 1 corresponds to the
electrooxidation of NADH.
Fig. 2
Effects of experimental parameters on the electrocat
alytic response of the ADH / PPSSWCNT / EPPG e
lectrode for the ethanol oxidation in 0. 05 mol·L - 1
TBS ( pH 8. 2) in the presence of 20 mmol·L - 1
ethanol
a. SWCNT loading, b. ADH loading, c. NAD +
concentration, d. pH in above cases, the anodic
peak current ( I p a ) values were estimated at 5 mV
·s - 1
Fig. 1
CVs obtained at the ADH / PPSSWCNT / EPPG e
lectrode in 0. 05 mol·L - 1 TBS ( pH 8. 2) contai
ning 10 mmol · L - 1 NAD + in the presence
( ———) and absence ( - - -) of 20 mmol · L - 1
ethanol
inset shows the voltammetric responses of 5 mmol
·L - 1 NADH at the bare EPPG (- - -) and PPS
SWCNT / EPPG ( ———) electrodes in 0. 05 mol·
L - 1 TBS ( pH 8. 2)
scan rate: 5 mV·s
3. 2
-1
Optimization of Electrocatalysis for
Ethanol Oxidation
The performance of the ADH / PPSSWCNT / EP
PG electrode towards the ethanol oxidation was found
to be depended on the loading amounts of SWCNT
and ADH, the NAD
+
concentration and pH ( Fig.
2) . As shown in Fig. 2a, the electrocatalytic current
increases with the increasing loading amount of SWC
NT and reaches a maximum value at 20 μL of SWC
NT . When the loading amount of SWCNT is higher
than 2 0 μL , the response remains same which means
that this amount is effectively enough to observe a suf
ficient response for the electrocatalysis of ethanol.
The magnitude of the current response of the am
perometric ethanol biosensor depends mainly on the
enzyme kinetics which determines the rate at which
NADH is generated enzymatically within the ADH /
PPSSWCNT biocomposite and the electrochemical re
action kinetics which controls the rate at which NADH
can be converted to NAD + to generate the measured
electric signal within the ADH / PPSSWCNT biocom
posite. Fig. 2b shows the effect of the amount of en
zyme dropped on the electrode surface on the voltam
metric response of the enzyme electrode. With in
creasing the loading of the ADHBSAGA mixture,
the catalytic peak current increases and the current
reaches an almost constant value at the loading of
more than 10 μL, indicating the presence of an ade
quate amount of enzyme in the PPSSWCNT modified
electrode. Thus 10 μL of the ADHBSAGA mixture
was used for preparation of the ethanol biosensor.
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Farhana S. Saleh &:'()*+,-./0123456789:;<=>?@A
· 267·
The NAD + concentration in the ethanol solution
is another important parameter for ethanol detection.
The effect of the NAD + cofactor concentration on the
response of the biosensor for 20 mmol·L - 1 ethanol
was investigated using the aboveoptimized enzyme
loading. The NAD + concentrations in the solution
ranged between 3 and 20 mmol · L - 1 , and the ob
tained results are displayed in Fig. 2c. The signal in
creases with the increase in the concentration of
NAD + up to 10 mmol · L - 1 and remains practically
constant at the higher concentration.
As the enzyme activity is dependent upon the pH
value of a buffer solution, the effect of the solution
pH of 0. 05 mol·L - 1 TBS containing 10 mmol·L - 1
ue in the range of 6. 0 to 8. 2, and reaches a maxi
Current ~ time response of the ADH / PPSSWCNT /
EPPG electrode for the successive addition of 250
-1
-1
μmol·L ethanol at 0. 2 V in 0. 05 mol · L
-1
+
TBS ( pH 8. 2) containing 10 mmol·L NAD
C ethanol / μmol·L - 1 : a. 250 , b. 500 , c. 750 ,
d. 1000, e. 1250, f. 1500, g. 1750,h. 2000,
i. 2250
the solution was stirred with a magnetic stirrer at
300 r / min
an increase in pH value above 8. 2. Therefore, a so
· L - 1 ethanol to the solution . In this case , the elec
NAD on the current response of 20 mmol·L
+
-1
etha
nol at the ADH / PPSSWCNT biocomposite film modi
fied EPPG electrode was examined. A plot of the cur
rent response against pH is shown in Fig. 2d. The
current response increases with increasing the pH val
mum value at pH 8. 2. It decreases dramatically with
Fig. 3
lution of pH 8. 2 and 10 mmol · L - 1 NAD + in 0. 05
trode potential was kept at 0. 2 V vs. Ag | AgCl | KCl
tivity of the ethanol biosensor.
current increased steeply to a stable value within 5 s,
mol·L
3. 3
-1
TBS were selected for examining the sensi
Amperometric Determination of Eth
anol
Based on the good electrocatalysis of the ADH /
PPSSWCNT / EPPG electrode for the ethanol oxidat
ion,its amperometric response as ethanol sensor was
examined in a stirred 0. 05 mol·L - 1 TBS ( pH 8. 2)
( at 300 r / min ) . After stabilization of the baseline
current, ethanol was injected into the buffer solution.
ADH catalyzes the oxidation of ethanol and simultane
ously the cofactor NAD + gets reduced to NADH ac
cording to the following enzymatic reaction.
ADH
Ethanol + NAD + →Acetaldehyde + NADH + H +
(1)
( sat. ) . Upon addition of an aliquot of ethanol, the
demonstrating that the electrocatalytic response is very
fast. The plot of the current vs. ethanol concentration
gave a good calibration graph as shown in Fig. 4. The
linear response range is upto ~ 1. 0 mmol · L - 1
which is found to be wider than the literature value
(10 ~ 425 μmol · L - 1 ) [30] . From the slope of the
linear portion, the sensitivity was calculated to be 2. 0
-2
-1
μA· cm · mmol · L and the limit of detection
( LOD) was estimated as 36 μmol·L - 1 . The LOD is
lower than that obtained by immobilizing ADH on Au
nanoparticles (49 μmol·L - 1 ) [31] and is much lower
than 0. 1 mmol·L - 1 and 90 μmol·L - 1 reported for
the sensors based on injection of the recognition ele
ment [32] and the immobilization of ADH on SWCNT
According to this reaction, the signal resulting
via polyelectrolyte of poly ( dimethyldiallylammonium
the concentration of ethanol. Fig. 3 shows the current
The operational stability of the ADH / PPSSWC
from the NADH oxidation increases with increasing
time response to the successive addition of 250 μmol
chloride) [33] , respectively.
NT / EPPG sensor was also examined by a continuous
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i. e. , the electrode fouling. In addition, a large sur
face area of the SWCNT allows a large amount of
ADH to be immobilized within the carbon nanotubes
assembly, resulting in a CNTsbased enzyme reser
voir.
4
Conclusions
A new kind of ethanol sensor based on the de
hydrogenase enzyme immobilized onto PPSfunction
alized SWCNT nanocomposite modified EPPG elec
trode was successfully constructed. The fabricated
ethanol biosensor provided a reasonable sensitivity of
Fig. 4
The plot of the steadystate current against ethanol
concentration using the data obtained from Fig. 3
measurement of 1. 0 mmol · L - 1 ethanol at the ap
plied potential of 0. 2 V over a period of ca. 45 min.
Fig. 5 shows a considerably stable amperometric re
sponse with a less than 7% signal drift demonstrating
a considerable resistance against the socalled elec
2 . 0 μA · cm - 2 · mmol - 1 · L and a low detection
limit ( 36 μmol · L - 1 ) with a linear concentration
dependence upto ~ 1 . 0 mmol · L - 1 at a detection
potential of 0 . 2 V.
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