iv>
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.
Copyright © 2012 SciRes. ENG
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/s120mv/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.
Copyright © 2012 SciRes. ENG
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.
REFERENCES
[1] JIANG Zhongfa, YUAN Wenjun, ZHANG Benyan. Progress of
study on the relationship between Indoor air formaldehyde
Pollution and Leukemia[J] , Journal of Environment and Health,
2008,25(3): 276-278 (in Chinese).
[2] ZHAI, Jinxia, ZHANG Chuanmu, ZHANG Peng. Acute effects
of formaldehyde in anatomy laboratory on student health[J].
Journal of Environment and Health, 2008,25(2): 141-143 (in
Chinese).
[3] Yang JZhang Bet a1Cloning and Expression of Pseudo-
monas fluorescens 26-2 lipase gene in Pichla paaoris and cha-
racterizing for transosterificationAppl Biochem Biotechnol
2008159(2)355-365
[4] Mitsubayashi K, NishioG, SawaiM, A bio-sniffer stick with
FALDH (formaldehyde dehydrogenase)for convenient analysis
of gaseous formaldehyde[J] . Sensors and ActuatorsB, 2008,
130( 1 ) : 32~ 37.
[5] Boukerma KPiquemal J YChehimi M Met alSynthesis
and interfacial properties of montmorillonite/polypyrrolenano-
compositesJ].Polym.,200647:569-576
[6] R en X ZZha o QLiu J Het alPrepa ration of polypyrrole
nanop-articles in reverse micelle anditsapplication to glucose
biosensorJ].Journal of Nan osc ienc e and Nanot echn olog y
doi:101166/jnn2007(2):141
[7] Wang S G,Zhang Q,Wang R L,et al.A Novel multi-walled car-
bonnanotube-based biosensor for glucose detection[J].Biochem
BiophysRes Commun,2003,3 11(3):572 ~576.
[8] ZHANG Shu-ping, ZHAO Yan, MAJie1, LIUXiao-hui,
WANGMeng-fei, LIUWei. Study on performance of
layer-by-layer self-assembled electrodein detection of thiocho-
line[J].Modern Chemical Industry,2011,Jan,31(1):88~92
[9] William G.Gutheil, Elvin Kasimoglu, and Permila C.Nicholson.
Induction of Glutathione-Dependent Formaldehyde Dehydroge-
nase Activity in Escherichia coli and Hemophilus influenza[J].
Biochemical and biophysical research communications 238,
693--696 (1997)
[10] William G.Gutheil,Barton Holmquist,and Bert L.Vallee. Purifi-
cation, Characterization, and Partial Sequence of the Gluta-
thione-Dependent Formaldehyde Dehydrogenase from Esche-
richia coli: A Class I11 Alcohol[J]. Biochemistry 1992, 31,
475-481.
[11] Shi yintao,Yuan ruo,Wang na, The biosensor based on polythio-
nine Nano Au and horseradish peroxidase modified for detecting
Hydrogen peroxide[J], Southwest China Normal Universi-
ty,2006,31,3.
[12] hou guoqing, The biosensor based on polyvinylpyrrolidone/Nano
Au and horseradish peroxidase modified for detecting Hydrogen
peroxide[J], S outhwest China Normal University,2010,36,4.
[13] M. Ben Ali, M. Gonchar, G. Gayda, S. Paryzhak, M.A. Maaref,
N. Jaffrezic-R enault , Y. Korp an . Forma ldehyde-sensitive sensor
based on recombinant formaldehydedehydrogenase using capa-
cit anc e versu s volt age mea su remen ts[ J] . Biosen sor s and Bioelec-
tronics 22 (2007) 2790–2795
[14] Kohji Mitsubayashi, Genki Nishio, Masayuki Sawai, Elito Ka-
zawa . A biochemical sniffer-chip f or conveni ent analysis of ga-
seousformaldehyde from timber materials[J]. Microchim Acta
(2008) 160: 427433.
[15] Lilach Bareket, Ada Rephaeli, Gili Berkovitch, Abraham Nu-
delman, Judith Rishpon. Carbon nanotubes based electrochemi-
cal biosen sor for detec tion of formaldeh yde rel eased from a ca n-
cer cell line treated with formaldehyde-releasing anticancer pro-
drugs[J]. Bioelectrochemistry 77 (2010) 9499.
[16] Yar oslav I. Korpan & Olexand r O. Soldatki n & Olga F. Sosovs-
ka & Halyna M. Klepach . Formaldehyde-sensiti ve conductome-
tric sensors b ased on commerc ial an d recombi nant formaldehyde
dehydrogenase[J]. M ic r o chim Acta (2 01 0) 170:33 7344.