Engineering, 2012, 5, 135-138
doi:10.4236/eng.2012.410B035 Published Online October 2012 (http://www.SciRP.org/journal/eng)
Formaldehyde Biosensor with Formaldehyde Dehydrogenase
Adsorped on Carbon Electrode Modified with Polypyrrole and
Carbon Nanotube
Mingwei Wa ng, Shuhai Jiang, Yi Che n, X i ang Chen, Li Zhao, J uankun Zhang *, Jinfe ng Xu
Key Laboratory of Industrial Microbiology, Ministry of Educati on, College of Biotech nology,
Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology,
Tianjin Universi ty of Science&Technology, Tia nj in, 30 0 457, C hi na
Received 2012
ABSTRACT
In this study, a carbon electrode electroless polymerization of polypyrrole, which formed a layer of conductive film interface,then
absorped a layer of carbon nanotube particles by the way of self-assembled, is studied. The modified electro d e is u sed to elect ro static
adherence of formaldehyde dehydrogenase, with Nafion solution capped. Moreover this paper discusses the work o f electrode po si-
tion, PH value and scanning speed on the influence of the electrode response, the response potential is -1.2v, the optimal PH value
is 7.5, and we also review the l inear r ange o f this electr od e, we disco vered t aht this biosensor have a good linear relationship with the
concentration of 1ug/ml-360ug/ml, the Correlation coefficient is 0.9983. In addition,with the stability of this electrode t ested, it can
be store d 15 d at 4°C.
Keywords: Formaldehyde Dehydrogenase; Eletrostatic Self-assemb ly; Biosensor
1. Introduction
Formaldehyde has been identified as carcinogenic and terato-
genic substances by the World Health Organization, and it is
the recognized source of allergy and a potentially strong muta-
gens, ranking second on the list of priority control of toxic
chemicals in china[1]. Formaldehyde on human health is main-
ly manifest ed in the abno rmal se nse of s mell, irritation, allergy,
pulmonary function abnormalities, abnormal liver function and
immune function abnormalities[2].
Making use of the catalytic properties of formaldehyde
dehydrogenase for detecting formaldehyde, is not only easy to
operate and specific, but also suitable for everyday use in the
workplace. For example, Vianello’s group[3] have coupled
formaldehyde dehydrogenase on field-effect transistor, to detect
airborne formaldehyde content by ion FET sensitive transistive,
and the detection limit is 0.1ug/ml, much lower than the critical
value of the occupational exposure. Mitsubavashi’s group[4]
have fixed for mal dehyde d eh ydrogena se on a p lati num electro d e,
to detect the production of NADH when enzyme reactions
happen, which can reflect the content of formaldehyde. In this
study, the l in ear ran ge is 4 0-3 0 0ug/ ml. M. Be n Ali ’s group [13]
manufactured a biosensor with formaldehyde dehydrogenase,
which was reco mbinated b y Genetic engin eering, and used this
biosensor to detect trace amounts of formaldehyde with
stripping voltammetry. Kohji. Mitsubayashi [14] developed a
convenient biochemical chip for testing wood materials in
gaseous formaldehyde. Lilach Bareket [15] developed a
electrochemical biosensor based on carbon nanotubes for
detecting formaldehyde release from cancer precursors drugs.
Yaroslav [ 16] fixed formald ehyde d ehydrogen ase and co factors
on the surface of electrode, which avoided adding coenzyme
and glutathione when detecting substrates, and the range of this
biosensor is 10mM—200mM.
In this study, we used the technology of electrostatic self-
assembly for sdsorbing enzyme. Firstly, we polymerized
polypyrrole on the surface of carbon electrode, Then we made
it adsorb carbon nanotubes.At last, we put it in the solution
cont aining formalde dehydrogenase, for adsorbing enzyme by
electrostatic self-assembly. By this way, we produced a new
and excellent biosensor for detecting formaldehyde.
2. Experiment
2.1. Reagents
Formaldehyde dehydrogenase was produced according to the
papers of[9,10]. Pyrrole is purchased from the company of ke
wei of tian jin.Carbon nanotubes was obtained in Shenzhen
Nanotech Port Co,Ltd. Potassium ferricyanide is purchased
from the company of Chemical Reagent of Yong Da in TianJin.
All other reagents like as Na2HPO4.12H2O, NaH2PO4.H2O,
NaCl, Anhydrous alcohol were of analytical grade.Pyrrole was
freshly distilled before use.Double distilled water was used for
the preparation of all buffer solutions.
2.2. Apparatus
A LK2005A model potentiostat from lan li ke in tianjin driven
by an PHILIPS PC with software was used for electropoly-
merization and detection measurements. A three—electrode
cell with a saturated Ag /AgCl reference electrode and aplati-
num foil counter electrode was used. Electronic analytical bal-

M. W. WANG ET AL.
136
ance is the model of FA2004A from the company of shanghai
Precision & Scientific instrument. A PH meter is from Shang-
hai Magnetic Technology Co.,Ltd. Ultrasonic cleaning instru-
ment is from Ningbo Chi Biotechnology Co.,Ltd.
2.3. Characteristics and Functions of Material and
Technology
Polypyrrole has been in-depth studyed because of its excellent
conductivity and simple synthetic process. It has been found
that various additives and doped or composite nanoparticles in
the polypyrrole ,not only improve the electrical conductivity of
polypyrrole,but also enhance its thermal stability and mechanic-
cal ductility. In this research,we use the method of electro-
chemical polymerization to form a polypyrrole film on the sur-
face of carbon electrode[5 ,6].
As a unique nano-materials,Carbon nanotubes has large
speci fic sur face area, excellent electric al,chemical properti es and
biological affinity. we can modify the carbon nanotubes through
replacing, addition and oxidation, whether at the surface, the
end or the tube, for introducing functional groups and bio-
active co mponen t. This can b e used for enz yme immobi lizatio n
materials, or be served as the base electro d e modified materials.
In one word, we made a new type of carbon nanotube modified
enzyme sensor[7].
Electrostatic self-assembly as a new biomolecular immobili-
zation method has been incr easingly used fo r the p reparation of
various biological sensors. With the principle of electrostatic
self-assembly of supramolecular, it can make positive and ne-
gatively charged substances (nanoparticles, dyes, polyion, DNA,
protein)attract in g each other, and this method has more advan-
tages considering biological activity, performance, stabil- ity,
simple preparation,and preparation under mild condi- tions [8].
2.4. Preparation of Enzyme Electrode[11,12]
Polish the carbon electrode with 0.05um Al2O3 suspension until
it looks like a mirror, rinse clean,and then ultrasonic clean in
distilled water and dilute sulfuric acid solution each with 10
minutes. Get out this processed eletrode,using the Three-elec-
trode system to active, under 0.4—﹣1.6v potential, with the
speed of 50mv/s, scan by cyclic voltammetry in 0.5mol/L
H2SO4 solution with 10 laps. After the activation electrode is
removed, rinsed, and dried at room temperature. Then put this
electrode into 0.1mol/L pyrrole solution scanning by cyclic
voltammetry with 60 laps, at the speed of 100mv/s. Put this
electrode modified with polyprrole into carbon nanotube solu-
tion for 12 hours, and then transfer it to Formaldehyde dehy-
drogenase solution for 24 hours under 4°c, wash by stilled wa-
ter, Finally drop nafion solution for capping,dry and put it in
PBS solution under 4°c for using.
2.5. The Preparation of the Substrate
In this study, all substrates for detecting contant 0.1mol/LPBS,
4.8mol/LNAD+, 1.0mol/L Glutathione,and with different con-
centr a t ion of formalde hyde.
2.6. The Mathods of Detection
The device of the three-electrode: the reference electrode is
Ag/AgCl, the counter electrode is platinum electrode, the
working electrode is carbon electrode modified by Formalde-
hyde dehydrogenase. Under the condition of experiment, we
use cyclic voltammetry and square wave voltammetry to test
with the PBS buffer solution at PH=7.0, and the scanning po-
tential is ranging from 0.4v to -1.6v at the scanning speed of
50mv/s. The current response of NADH is record by the en-
zyme electrode. When the enzyme electrode is not used,stored
it in PBS buf fer solut io n u nde r 4°c at the PH value of 7.0.
3. Results and Discussion
3.1. The Preparation of Polypyrrole Film
Cyclic voltammetry can make a very uniform film with ele-
troless polymerization. The cyclic voltammogram of polypyr-
role modified carbon eletrodes is showed in Figure 1, the po-
tential is between 0.4v and -1.6v, scanning speed is 100mv/s,
the concentration of pyrrole is 0.1mol/L with 60 laps. Accord-
ing to this picture, there are two obvious reduction peak in near
-0.42 v and -0.78v, and also a oxidation peak in -0.42v, with the
gradual increase of the aggregate number of laps. Reduction
peaks and oxidation peaks in varying degrees are reduced, in-
troducing the conductive properties declined with the polypyr-
role increased,and last tend to stability,but the conductivity is
also exist.
3.2. The Determination of the Test Potential
In order to find out the potential of NADH in reactions, we
used square wave voltammetry to test in PBS solution and sub-
strate with fo r maldeh yde an d o ther acc esso r y facto rs. In Figure
2, there are two lines ,the line with one peak is the modified
electrode in PBS solution,the other is in the substrate. Both of
this lines have the same reduction peak in -0.6v, whic h is be-
cause of PBS. The second line has two other peaks:-1.2v and
-1.5v, in substrates with different concentration formaldehyde.
The peak of -1.2v is changed, and the peak of -1.2v is stable, so
we can see the peak can show the speed of production of
NADH.The peak in -1.5v is not known of its mechanism.
Figure 1. Electrochemical Polymerization of polypyrrole.

M. W. WANG ET AL.
137
3.3. The Influence of Sc anning Spe e d
In the solution of substrate with 180ug/ml formaldehyde, we
used cyclic vol tammetr y to scan t he potential between 0.4v and
-1.6v, with the speed from 50mv/s—120mv/s. From the Figure
3, we can see that, when the speed exchange, only the current
peak increased, the potential peak is stable at nearly -1.2v, so
we can sa y that the reaction is subj ect to the control of surface.
3.4. The Electrochemical Response of the Sensor on
Formaldehyde
In th is reseach, we detect ed different substrates with concentra-
tion: 50ug/ml, 20ug/ml, 15ug/ml, 10ug/ml, 6ug/ml, 1ug/ml, and
responds were 22.967uA, 11.946uA, 10.542uA, 8.397uA,
6.534uA, 4.628uA. From the Fig ure 4, the sensor to different
concentration of formaldehyde shows a good liner relationship,
and its standard curve equation is Y=0.3717X+4.5169, with
correlation coefficient of 0.9983 . The detection limit is
0.1ug/ml, and the detection ranges from 0.1ug/ml to 360ug/ml.
Figure 2. Testing in PBS solution and substrate of formaldehyde.
Figure 3. Different scanning speed for the electrode.
3.5. The Influence of PH
Formaldehyde dehydrogenase catalyzed formaldehyde to for-
mic acid ,wh ich makes the solu tion PH value decline. However,
the concen tration of for maldehyde det ected in the experi mentl y
is trace, an d formic acid is a wea k acid , so th e P H valu e chan ge
is extre mely small in reaction, it can be ignored . The only thing
we must care about is the PH value of initial reaction substrate.
In Figure 5, we used square wave voltammetry to detect re-
sponse of enzyme electrode current in 180ug/ml substrate,when
other conditions excpt PH is unchanged. From the picture, we
can see th at the best respon se value of this eletr ode is 7.5, indi-
cating that formaldehyde dehydrogenase optimum PH value is
7.5.
3.6. Stability Test of Enzyme Electrode
Put the enzyme eletrode at 4°c in PBS solution, using square
wave volt ammetry to d etect su bstrat e in th e same concent ratio n
of formaldehyde at different times. From the pictrue of Figure
6, is the conclusion of the test at 1day, 10days,15days, 20days,
Figure 4. The liner response of different concentration range.
Figure 5. I nfluence of the Sensor by the PH value .

M. W. WANG ET AL.
138
Figure 6. Testing at different times for stability.
25days, 30days, 40days, 45days, we can see that:the enzyme
electrode at 1day, 10days, 15days is no obvious differece for
testing the same substrate, but after 20days, the current is ob-
vious reduced and has deviated from the standard curve, so the
retention time of this electrode in 4°c environment is 15 days.
4. Conclusions
Using polypyrrole as matrix, with the principle of electrostatic
adsorption, we make the biosensor modified by enzyme for
deteacting formaldehyde. In the biosensor,carbon nanotubes
and formaldehyde dehydrogenase are immobilized on the sur-
face of carbon eletrode, and nafion solution capped to prevent
leakage of carbon nanotubes and enzyme. The sensor with for-
maldehyde dehydrogenase modified is simple and easy ,with a
good linear range, and also have practical value. However,
there is some room for improvement. If we can fix NAD+ and
glutathione with formaldehyde dehydrogenase on the surface of
electrode, we don’t need to add them for test each time. The
purity of enzyme solution is also need to improve, which may
increase t he response range of the substr ate concentr ation.
5. Acknowledgements
The authors would like to thank the Ministry of Science and
Technology of the People’s Republic of China (2009GJA10047),
Tianjin Municipal Science and Technology Commission
(09ZCZDSFO4200), Tianjin Municipal Education Commission
(SB20080035) and the Tianjin University of Science and
Technology to support the work.
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