Template-free enzymatic synthesis of electrically conducting polyaniline using soybean peroxidase

EUROPEAN

European Polymer Journal 41 (2005) 1129–1135

www.elsevier.com/locate/europolj

POLYMERJOURNAL

Template-free enzymatic synthesis of electrically conductingpolyaniline using soybean peroxidase

Rodolfo Cruz-Silva, Jorge Romero-Garcıa *, Jose Luis Angulo-Sanchez,Antonio Ledezma-Perez, Eduardo Arias-Marın, Ivana Moggio,

Erika Flores-Loyola

Departamento de Biopolımeros, Centro de Investigacion en, Quımica Aplicada (CIQA),

Blvd. Enrique Reyna 140, Saltillo, Coahuila 25100, Mexico

Received 22 June 2004; received in revised form 4 October 2004; accepted 12 November 2004

Available online 13 January 2005

Abstract

Synthesis of polyaniline (PANI) catalyzed by soybean peroxidase at 1 �C in either aqueous or partially organic

media, was studied as a function of pH and reaction media. Kinetic studies indicated that, unlike chemical polymeri-

zation, enzymatic polymerization of aniline showed neither induction period nor auto acceleration. The redox revers-

ibility and chemical structure of the synthesized PANI was strongly dependent on the starting pH of the reaction

medium. UV–vis, FT-IR, WAXD and TGA analysis are used to explain how the enzymatic reaction conditions influ-

ence both the chemical structure and physical properties of the PANI. Optimal reaction conditions are outlined for the

direct enzymatic synthesis of electrically conductive emeraldine salt with yield as high as 71%.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Polyaniline; Enzymatic polymerization; Soybean peroxidase; Conducting polymers

1. Introduction

Polyaniline (PANI) is an intrinsically conductive

polymer, which has been widely studied because of its

high chemical and thermal stability, good electrical con-

ductivity and broad potential applications [1]. The com-

mon method used for synthesize PANI is by chemical

oxidation of aniline. However, the major drawbacks of

this method are their harsh synthetic conditions, because

chemical oxidation is carried out in extreme acidic reac-

0014-3057/$ - see front matter � 2004 Elsevier Ltd. All rights reserv

doi:10.1016/j.eurpolymj.2004.11.012

* Corresponding author. Tel.: +52 844 438 9830; fax: +52

844 438 9839.

E-mail address: [email protected] (J. Romero-Garcıa).

tion media using a large amount of strong oxidizing

agent, such as ammonium persulfate [2]. During the oxi-

dation of aniline a large amount of sulfonium anion and

ammonium sulfate salt are released as by-products. This

anion competes in the doping process of PANI, thus the

synthesized PANI contains traces of sulfate as contami-

nant [3].

During the last decade, enzymatic polymerization of

aniline has attracted great attention because it is carried

out under milder conditions, in comparison with those

used for chemical polymerization. Horseradish peroxi-

dase (HRP) and soybean peroxidase (SBP) are oxidore-

ductase enzymes capable of oxidize aromatic amines in

the presence of hydrogen peroxide [4–6]. These enzymes

are derived from non-contaminant renewable sources,

ed.

Table 1

Electrical conductivity and yield of PANI synthesized by

1130 R. Cruz-Silva et al. / European Polymer Journal 41 (2005) 1129–1135

have high reaction selectivity to aromatic compounds

and cut down the oxidation by-products to water [7].

For these reasons, peroxidase catalyzed polymerization

of aniline has been considered an environmentally

friendly route to obtain PANI.

Most of the works regarding enzymatic polymeriza-

tion of aniline have been done using polyelectrolyte

templates such as sulfonated polystyrene [8–10],

poly(vinylphosphonic acid) [11] and deoxyribonucleic

acid (DNA) [12], among others. Such studies have

shown that these templates provide a local environment,

which promotes para-directed, head-to-tail coupling of

aniline radicals. This polyanion-assisted polymerization

allows the enzymatic syntheses of water-soluble com-

plexes of conducting PANI with well-defined structure

at pH as high as 4.3 [13]. However, the high degree of

complexation between PANI and the polyanion makes

difficult to obtain bulk polyaniline, which is required

for specific applications, such as free standing films

[14] or fibers [15].

Zemel and Quinn [16] reported that bulk electrically

conductive polyaniline can be synthesized using HRP

as catalyst, in aqueous media at pH 3.0. However, under

such reaction conditions, the HRP go through a rapid

denaturing process, thus to carry out the reaction large

amounts of this enzyme are needed. The activity loss

can be avoided performing the reaction at pH above

6.0 in aqueous media [17] but, when polyaniline synthe-

sis is carried out at high pH in absence of an anionic

template, a non-conductive material is obtained [18].

The lack of electrical conductivity is attributed to struc-

tural defects, such as branched or cross-linked chains

and phenyl–phenyl couplings, which causes loss of elec-

tronic conjugation of the PANI backbone.

SBP is an enzyme with higher thermodynamic stabil-

ity than HRP [19], as well as better catalytic activity un-

der acidic environment [20–22]. In spite of that, there are

not reports about the use of this enzyme for electrically

conducting PANI synthesis. In this work, we focused on

the study of the reaction conditions required to synthe-

size bulk conductive PANI catalyzed by SBP in a tem-

plate-free system. UV–vis, FT-IR, WAXD and electric

conductivity studies revealed that PANI obtained by

enzymatic polymerization was the emeraldine salt form

with structural features quite similar to that chemically

synthesized.

Run 1,4-dioxane

( % v/v )

pH0 Yield (%) Conductivity

(S/cm)

1a 0 �1.0 74 5.4

2b 0 3.0 71 2.4

3b 30 3.0 39 6.4E-02

4b 0 5.0 19 <1E-06

5b 30 5.0 11 <1E-06

a C-PANI.b Enzymatically synthesized PANI using SBP.

2. Experimental

2.1. Materials

Aniline and ammonium hydroxide were obtained

from Quımica Dinamica, Mexico. Aniline was distilled

under reduced pressure over stannous chloride and

potassium hydroxide. The middle fraction was collected

and stored at �28 �C prior to use. Hydrogen peroxide

(30% wt.), 1,4-dioxane, N-methyl-2-pyrrolidinone

(NMP) and hydrochloric acid (HCl) were purchased

from Aldrich. 4-Toluensulfonic acid (TSA) was pur-

chased from Merck. Horseradish peroxidase (HRP)

(Type II, 200 U/mg) and soybean peroxidase (SBP)

(64 U/mg) were obtained from Sigma and used as

received.

2.2. Polyaniline synthesis

Chemical via: chemically synthesized polyaniline (C-

PANI) (run 1, Table 1) was obtained according to the

procedure reported by Wei and Hsueh [23], and used

as reference material. C-PANI was converted to its base

form by treatment with an excess of aqueous NH4OH

(0.1 M) for 2 h, then was washed with distilled water

and freeze-dried.

Enzymatic via: enzymatic reactions (runs 2–5, Table

1) were carried out in aqueous or partially organic reac-

tion medium (dioxane/water, 30:70 volume). Typically,

aniline (1.83 g) was dissolved in 60 ml of reaction med-

ium. The starting pH (pH0) was adjusted to 3.0 or 5.0

by adding a 25 wt% TSA solution. Afterwards, the reac-

tion mixture was degassed for 20 min by alternating vac-

uum/nitrogen purges and cooled to 1 �C. Then, SBP or

HRP was added to reach a 0.5 mg/ml concentration, fol-

lowed by stepwise addition (80 ll each 12 min) of hydro-

gen peroxide until a 1:1 molar ratio to the aniline was

reached. The reaction was kept in agitation and under

continuous nitrogen purge for 8 h to complete the poly-

merization. Finally, the precipitated material was col-

lected by filtration, washed successively with methanol

and an aqueous solution of TSA (5% w/v), and freeze-

dried.

2.3. Instruments and methods

Fourier transformed infrared (FT-IR) spectra were

measured in KBr pellets on a Nicolet Magna 550 FT-

IR spectrophotometer. UV–vis electronic absorption

spectra of PANI solutions were recorded on a Hew-

0 10 20 30 40 50 601.8

2.2

2.6

3.0 hydrogen peroxide addition

SBP addition

pH

Time (min)

Fig. 1. pH change versus time during the enzymatic synthesis of

PANI carried out in aqueous media at pH0 of 3.0. The arrow

pointing down indicates SBP addition and the arrows pointing

up indicate each addition of 80 lL of hydrogen peroxide

(30 wt%).

R. Cruz-Silva et al. / European Polymer Journal 41 (2005) 1129–1135 1131

lett–Packard 8452A UV–vis spectrophotometer using

NMP as solvent. Thermogravimetric analyses (TGA)

were conducted under a nitrogen flow (50 ml/min) at a

heating rate of 20 �C/min in a TA Instruments 2920

equipment. Prior to thermal analysis, polymers were

dedoped and dried in an oven at 50 �C. Real-time pH

measurements were done using a pH/ion-meter Dow

Corning 450 coupled to a PC. Wide angle X-ray diffrac-

tion patterns (WAXD) were collected on a Siemens D-

5000 X-ray diffractometer with a CuKa radiation

source, (intensity 25 mA, acceleration voltage 35 kV).

The data were collected in the 2h mode with a scan rate

of 0.3 �/min at room temperature. For electrical conduc-

tivity measurements PANI materials were redoped. In

the case of enzymatically synthesized dedoped PANI,

it was suspended overnight in a 10% w/w solution of

TSA, while the dedoped C-PANI was treated overnight

with a 1.0 N HCl solution. The redoped powders were

filtered on a Buchner funnel and freeze dried. Electrical

conductivity measurements were done according to the

two-probe technique using a digital multimeter from Ex-

tech Instruments. The electrical resistance of cylindrical

compressed pellets (5 mm diameter and 7.5 mm height)

of PANI powder were converted to electrical conductiv-

ity using Ohm�s Law. To minimize contact resistance,

the bottom and top surfaces of the pellets were covered

with a thin layer of conductive silver paint.

3. Results and discussion

3.1. Synthesis

When the enzymatic polymerization of aniline was

carried out at pH0 of 3.0, either in aqueous or partially

organic medium, (runs 2 and 3, Table 1) a color change

to dark green within a minute after the first peroxide

addition was developed, which is indicative of emeral-

dine salt formation [24]. Reactions carried out at pH0

of 5.0, either in aqueous or partially organic medium,

(runs 4 and 5, Table 1) also developed a rapid color

change, but in this case to black. Fig. 1 shows the pH

change during the first hour of aniline polymerization

at pH0 of 3.0 in aqueous medium (run 2). The addition

NH3 TSA + H2O

NH

NH

NH

NH

TSA

TSA

4 2

Scheme 1

of SBP was followed by a short time to allow its disso-

lution. Fig. 1 shows that immediately after each addition

of hydrogen peroxide a decrease of the pH was regis-

tered. This pH change may be attributed to the release

of TSA, which at the beginning of the reaction is present

as the aniline counter ion, but once the reaction takes

place, is released from the partially doped polymer, as

it is depicted in the Scheme 1. The pH at the end of

the reaction was about 1.7, that is close to the pH limit

where SBP still shows catalytic activity [22]. The highest

polymer yield was obtained using SBP in aqueous med-

ium at pH0 of 3.0 (71%). Although we also observed a

rapid color change to green when HRP was used under

the same reaction conditions, the yield of only 25% indi-

cates that apparently HRP losses its catalytic activity at

higher rate than SBP. These results are in agreement

with the still good catalytic activity reported for SBP,

at pH value as low as 2.0 [21], while the HRP rapidly

inactivates at pH below 3.0 [25].

It is worthy to mention that enzymatic oxidation of

aniline did not present an induction period, as the ob-

served in C-PANI synthesis. This induction period is

due to the autocatalytic mechanism of the chemical oxi-

dation of aniline [26] while in the enzymatically cata-

lyzed oxidation the free radical formation follows a

2

+ 2 H3O++ 2 TSA4+ H2O

SBP

.

1132 R. Cruz-Silva et al. / European Polymer Journal 41 (2005) 1129–1135

different mechanism [4], which is independent of the

PANI concentration.

3.2. FT-IR analysis

Fig. 2 shows the FT-IR absorption spectra of PANI

synthesized at different pH0 and reaction conditions.

The spectrum of enzymatically synthesized PANI in

aqueous medium, at pH0 of 3.0 (Fig. 2b), is quite similar

to that of C-PANI (Fig. 2a). In both spectra, the charac-

teristic bands of PANI are observed, such as the ring

stretching of the quinoid diimine and the benzenoid dia-

mine units at 1598 cm�1 and 1500 cm�1, respectively

[27,28]. Other absorption peaks are the weak C–N

stretching vibration at 1375 cm�1 due to the Q-B units

and the 1305 cm�1 peak due to C–N stretching in B

units. The aromatic C–H in-plane bending origins a

peak at 1170 cm�1 and the 828 cm�1 peak corresponds

to C–H out of plane bending. The position of the latter

has been assigned to two adjacent hydrogens in the aro-

matic ring indicating a 1,4-disubstituted ring [28,29].

The two peaks that appear at 1010 and 1030 cm�1, as

well as the peak at 696 cm�1 are most likely due to

S@O stretching [30] originated by the TSA traces in

enzymatically synthesized PANI, which were not possi-

ble to remove during dedoping treatment.

When PANI was enzymatically synthesized in aque-

ous medium, at pH0 of 5.0 (Fig. 2d) or in partially or-

ganic medium at pH0 of 3.0 (Fig. 2c), the spectra

resembled to that previously described, but a decrease

in the intensity of the peaks at 1598 cm�1 and

828 cm�1 was observed. Moreover, the peak arising at

1560 cm�1 in the PANI enzymatically synthesized at

pH0 of 5.0, is associated to C–C stretching in benzenoid

1800 1500 1200 900 600

(d)

(c)

(b)

(a)

Tran

smitt

ance

(a.u

.)

Wavenumber (cm-1)

Fig. 2. FT-IR spectra of (a) C-PANI, and enzymatically

synthesized PANI under different conditions: (b) aqueous

medium at pH0 of 3.0; (c) partially organic medium at pH of

3.0; (d) aqueous medium at pH0 of 5.0.

rings with a substitution pattern different from those of

linear PANI [17]. These results indicate that at higher

pH or in partially organic reaction media the formation

of branched or cross-linked polymers are favored, as it

has been suggested [18].

3.3. Thermal analysis of PANI

The thermogravimetric analysis of C-PANI and

enzymatically synthesized PANI under different reaction

conditions is shown in Fig. 3. The initial weight loss at

temperatures below 150 �C observed for all the analyzed

samples, can be attributed to residual water retained

after drying. Under the same drying conditions, C-PANI

(Fig 3a) and PANI enzymatically synthesized in aqueous

medium at pH0 of 3.0 (Fig. 3b) showed lower water

affinity than PANI synthesized at pH0 of 5.0 in aqueous

media or partially organic media (Fig. 3c and d, respec-

tively). The major weight loss of C-PANI occurred

above 400 �C (Fig. 3a) and it was ascribed to thermal

decomposition [23]. Although FT-IR and UV–vis analy-

sis showed remarkable similarity between C-PANI and

PANI enzymatically synthesized in aqueous medium at

pH0 of 3.0, the latter showed a lower thermal stability.

Residual TSA and chain defects may have promoted

degradation of PANI during heating, as it has been sug-

gested in others works [3]. Nevertheless, PANI enzymat-

ically synthesized in aqueous medium at pH0 of 3.0

showed similar degradation pattern than C-PANI and

almost 80 wt% of the sample remains at 600 �C, which

is a fairly good thermal stability.

3.4. UV–vis spectra

The electronic absorption spectra of PANI enzymat-

ically synthesized in aqueous medium at pH0 of 3.0 and

C-PANI are given in Fig. 4a and b, respectively. Both

0 100 200 300 400 500 60060

70

80

90

100

a) b) c) d) W

eigh

t los

s (%

)

Temperature (º C)

Fig. 3. TGA scans of (a) C-PANI, and enzymatically synthe-

sized PANI under different reaction conditions: (b) aqueous

medium at pH0 of 3.0; (c) partially organic medium at pH0 of

3.0; (d) aqueous medium at pH0 of 5.0. All polymers were

dedoped before analysis.

300 400 500 600 700 800

a) b) c) d)

Abso

rban

ce (A

.U.)

Wavelength (nm)

Fig. 4. UV–vis absorption spectra of (a) C-PANI, and enzy-

matically synthesized polyaniline under different conditions: (b)

aqueous medium at pH0 of 3.0; (c) aqueous medium at pH0 of

5.0; (d) partially organic medium at pH of 5.0.

5 15 25 35 45

c)

b)

a)

Inte

nsity

(a.u

.)

Diffraction angle (2θ )

Fig. 5. WAXD patterns of (a) C-PANI, and enzymatically

synthesized PANI at pH0 of 3.0 in: (b) aqueous medium or

(c) partially organic medium.

R. Cruz-Silva et al. / European Polymer Journal 41 (2005) 1129–1135 1133

spectra show two absorption bands. The first one at

330 nm (band I) is associated with the p–p* transition

of the aromatic rings, while the peak at 630 nm (band

II) has been assigned to a benzenoid to quinoid excitonic

transition [27,28]. The UV–vis spectrum of PANI enzy-

matically synthesized in partially organic medium at

pH0 of 3.0 was undistinguishable from that of PANI

enzymatically synthesized in aqueous media at the same

pH0 and it was omitted from Fig. 4 for clarity purposes.

In the spectra of PANI synthesized enzymatically at pH0

of 5.0 either in aqueous or partially organic medium

(Fig. 4c and d, respectively) the intensity of band II is

diminished, suggesting a lower quinoid units formation

[27]. In addition, a broad absorption between 400 and

500 nm was observed, this absorption has been associ-

ated to branched or cross-linked polyaniline [8,9,18].

The low quinoid units formation in the PANI synthe-

sized under these reaction conditions is also supported

by the FTIR analysis, where a decrease in the intensity

on the peak related to the quinoid units ( 1598 cm�1,

Fig. 2d) was observed.

3.5. Wide angle X-ray diffraction

The Wide angle X-ray diffraction patterns of the sam-

ples are given in Fig. 5. Both, C-PANI (Fig. 5a) and

PANI enzymatically synthesized in aqueous media at

pH0 of 3.0 (Fig. 5b) exhibits several diffraction peaks

at 2h of 9.3�, 15.3�, 21� and 25.5�, these peaks are char-

acteristic for the crystalline phase of the emeraldine salt,

referred in literature as ES-I [31]. However, when the

enzymatic synthesis was carried out in partially organic

media (Fig. 5c) at the same pH0, a marked decrease of

the crystalline fraction was observed. Although UV–vis

spectroscopy showed that both enzymatically synthe-

sized samples have the same effective conjugation, FT-

IR analysis indicates that minor differences between

the samples are present. Thus, the lower crystalline de-

gree might be a result of a small amount of defect sites,

such as ortho couplings, head-to-head couplings or

branchings, which reduce the chain packing capability

due to steric hindrance. This behavior is quite analog

to that of PANI containing bulky molecules diffused

into the polymer matrix [32].

3.6. Electrical conductivity

Table 1 summarizes the electrical conductivity data of

C-PANI and enzymatically synthesized PANI under dif-

ferent reaction conditions. PANI enzymatically synthe-

sized in aqueous medium, at pH0 of 3.0 showed an

electrical conductivity in the same order of magnitude

than those observed for C-PANI [23] or chemically syn-

thesized PANI using TSA as doping agent [33]. When the

enzymatic syntheses of PANI were carried out at pH0 of

5.0 (runs 4 and 5 in Table 1), the polymers obtained were

not conductive. Similar results have been reported for

chemically synthesized polyaniline at pH0 of 5.0 [34].

Furthermore, we found that the inclusion of 1,4-dioxane

in the reaction medium decreases the conductivity of the

PANI, synthesized at pH0 of 3.0 in almost two orders of

magnitude. This effect could be related to the appearance

of defects in the polymer backbone as discussed in the

WAXD section, because a less crystalline PANI has

lower interchain interactions and consequently, a re-

duced electron transport that affects its electrical conduc-

tivity, in a similar way to polyanilines with bulky groups

attached to the polymer backbone [35].

3.7. Further discussion

The influence of the reaction media in the structure of

the enzymatically synthesized PANI are in agreement

1134 R. Cruz-Silva et al. / European Polymer Journal 41 (2005) 1129–1135

with the work of Adams et al. [36], who found that low

temperature, high dielectric constant of the reaction

medium and a long and steady oxidation of the mono-

mer were important to avoid site defects in chemically

synthesized PANI. The kinetic study shown that the

enzymatic oxidation of aniline seems to proceed rapidly

and in independent stages either in aqueous or partially

organic media. Furthermore, the inclusion of 1,4-diox-

ane in the reaction medium reduce its dielectric constant,

due to a lower dipole moment of the 1,4-dioxane com-

pared to that of the water. Both factors lead to more site

defects in the PANI enzymatically synthesized as com-

pared to the C-PANI. Although the enzymatic synthesis

of PANI is carried out in a reaction medium with less

chemical species than the chemical synthesis, FT-IR

analysis showed traces of TSA. Both defect sites and

residual TSA in the enzymatically synthesized PANI

may have been responsible of its lower thermal stability

as compared to C-PANI.

4. Conclusions

Soybean peroxidase (SBP) is an effective biocatalyst

for aniline polymerization either in partially organic

or aqueous media using hydrogen peroxide as oxidiz-

ing agent. UV–vis, FT-IR spectroscopy and WAXD

studies indicate that PANI enzymatically synthesized

in oxygen-free aqueous medium at pH0 of 3.0 and low

temperature (1 �C) had a chemical structure and elec-

trical conductivity quite similar to C-PANI. Low yield-

ing, branching and ortho-coupling were promoted

when PANI was enzymatically synthesized in par-

tially organic medium or at pH0 higher than 3.0.

Since the rate of enzymatic oxidation of aniline shows

neither induction period nor auto acceleration, this

method may offer a much better control of the kinetics

of polymerization. The use of SBP, which is a better acid

resistant peroxidase compared to HRP, improves the

yielding of the reaction and reduces the amount of en-

zyme required for aniline oxidation. Since no anions

are released during oxidation, the reaction conditions

are milder compared to those of the conventional chemi-

cal route.

Acknowledgments

This work was supported by CONACYT through

the Research Grant J-38753-U, R. Cruz-Silva and E.

Flores-Loyola acknowledges CONACYT for the

scholarships provided. Authors acknowledge Eduardo

Cruz for technical assistance with real-time pH

measurements.

References

[1] MacDiarmid AG. Polyaniline and polypyrrole: where are

we headed? Synth Met 1997;84(1–3):27–34.

[2] Lux F. Properties of electronically conductive polyaniline:

a comparison between well-known literature data and

some recent experimental findings. Polymer 1994;35(14):

2915–36.

[3] Milton AJ, Monkman AP. A comparative study of

polyaniline films using thermogravimetric analysis and IR

spectroscopy. J Phys D: Appl Phys 1993;26(14):1468–74.

[4] Kobayashi S, Uyama H, Kimura S. Enzymatic polymeri-

zation. Chem Rev 2001;101:3793–818.

[5] Arias-Marin E, Romero J, Ledezma-Perez AS, Kniajansky

S. Enzymatic mediated polymerisation of functional ani-

line derivatives in nonaqueous media. Polym Bull 1996;37:

581–7.

[6] Mejias L, Reihmann MH, Sepulveda-Boza S, Ritter H.

New polymers from natural phenols using horseradish or

soybean peroxidase. Macromol Biosci 2002;2(1):24–32.

[7] Gross RA, Kumar A, Kalra B. Polymer synthesis by in

vitro enzyme catalysis. Chem Rev 2001;101:2097–124.

[8] Liu W, Kumar J, Senecal KJ, Samuelson L. Enzymatically

synthesized conducting polyaniline. J Am Chem Soc 1999;

121(1):71–8.

[9] Samuelson LA, Anagnostopoulos A, Alva KS, Kumar J,

Tripathy SK. Biologically derived conducting and water

soluble polyaniline. Macromolecules 1998;31:4376–8.

[10] Nabid RM, Entezami AA. Enzymatic synthesis and

characterization of a water-soluble, conducting poly

(o-toluidine). Eur Polym J 2003;39(6):1169–75.

[11] Nagarajan R, Trypathy SK, Kumar J, Bruno FF, Sam-

uelson LA. An enzymatically synthesized conducting

molecular complex of polyaniline and poly(vinylphos-

phonic acid). Macromolecules 2000;33(13):9542–7.

[12] Nagarajan R, Liu W, Kumar J, Tripathy SK, Bruno FF,

Samuelson LA. Manipulating DNA conformation using

intertwined conducting polymer chains. Macromolecules

2001;34(26):3921–7.

[13] Liu W, Cholli AL, Nagarajan R, Kumar J, Trypathy SK,

Bruno FF, et al. The role of template in the enzymatic

synthesis of conducting polyaniline. J Am Chem Soc

1999;121(49):11345–55.

[14] Cruz-Silva R, Romero-Garcıa R, Angulo-Sanchez JL,

Flores-Loyola E, Farıas MH, Castillon FF, et al. Com-

parative study of polyaniline cast films prepared from

enzymatically and chemically synthesized polyaniline.

Polymer 2004;14:4711–7.

[15] Wang HL, Romero RJ, Mates BR, Zhu Y, Winokur MJ.

Effect of processing conditions on the properties of high

molecular weight conductive polyaniline fiber. J Polym Sci

Part B: Polym Phys 2000;38(1):194–204.

[16] Zemel H, Quinn JF. US Patent 5,420,237. 1995.

[17] Akkara JA, Kaplan DL, Kurioka H, Uyama H, Kobay-

ashi S. Characterization of polyaniline synthesized by

horseradish peroxidase. ACS Polymeric Materials Science

& Engineering: Conference Proceedings 1996;74:39–40.

[18] Lim CH, Yoo YJ. Synthesis of ortho-directed polyaniline

using horseradish peroxidase. Process Biochem 2000;36(3):

233–41.

R. Cruz-Silva et al. / European Polymer Journal 41 (2005) 1129–1135 1135

[19] McEldoon JP, Dordick JS. Unusual thermal stability of

soybean peroxidase. Biotechnol Prog 1996;12(4):555–8.

[20] Nissum M, Schiødt CB, Welinder KG. Reactions of

soybean peroxidase and hydrogen peroxide pH 2.4–12.0,

and veratryl alcohol at pH 2.4. Biochim Biophys Acta

2001;1545(1–2):339–48.

[21] Wang B, Li B, Cheng G, Dong S. Acid-stable ampero-

metric soybean peroxidase biosensor based on a self-

gelatinizable grafting copolymer of polyvinyl alcohol and

4-vinylpyridine. Electroanalysis 2001;13(7):555–8.

[22] Blinkovsky AM, Dordick JS, Arnold JM, McEldon JP.

Peroxidase-catalyzed polymerization and depolymerization

of coal in organic solvents. Appl Biochem Biotechnol 1994;

49:153–64.

[23] Wei Y, Hsueh KF. Thermal analysis of chemically

synthesized polyaniline and effects of thermal aging on

conductivity. J Polym Sci Part A: Polym Chem 1989;

27(13):4351–63.

[24] Stejskal J, Spirkova M, Kratochvil P. Polyaniline disper-

sions 4. Polymerization seeded by polyaniline particles.

Acta Polym 1994;45(5):385–8.

[25] Chattopadhyay K, Mazumdar S. Structural and confor-

mational stability of horseradish peroxidase: effect of

temperature and pH. Biochemistry 2000;39:263–70.

[26] Chakraborty M, Mukherjee DC, Mandal BM. Dispersion

polymerization of aniline in different media: a UV–visible

spectroscopic and kinetic study. Langmuir 2000;16(6):

2482–8.

[27] Wei Y, Hsueh KF, Jang GW. A study of leucoemeraldine

and effect of redox reactions on molecular weight of

chemically prepared polyaniline. Macromolecules 1994;

27(2):518–25.

[28] Kulkarni MV, Viswanatah AK, Marimuthu R, Seth T.

Synthesis and characterization of polyaniline doped with

organic acids. J Polym Sci Part A: Polym Chem 2004;42(8):

2043–9.

[29] Ping Z, Nauer GE, Neugebauer H, Theiner J, Neckel A.

Protonation and electrochemical redox doping processes of

polyaniline in aqueous solutions: investigations using

in situ FTIR–ATR spectroscopy and a new doping system.

J Chem Soc Faraday Trans 1997;93(1):121–9.

[30] Lu X, Ng HY, Xu J, He C. Electrical conductivity of

polyaniline–dodecylbenzene sulphonic acid complex: ther-

mal degradation and its mechanism. Synth Met 2002;

128(2):167–78.

[31] Pouget JP, Josefowicz ME, Epstein AJ, Tang X, MacDi-

armid AG. X-ray structure of polyaniline. Macromolecules

1991;24(3):779–89.

[32] Winokur MJ, Mattes BR. Structural studies of halogen

acid doped polyaniline and the role of water hydration.

Macromolecules 1998;31(23):8183–91.

[33] Kulkarni MV, Viswanath AK. Comparative studies of

chemically synthesized polyaniline and poly(o-toluidine)

doped with p-toluene sulphonic acid. Eur Polym J

2003;40(2):379–84.

[34] Gospodinova N, Mokreva P, Terlemezyan L. Chemical

oxidative polymerization of aniline in aqueous medium

without added acids. Polymer 1993;34(11):2438–9.

[35] Wei Y, Hariharan R, Patel SA. Chemical and electro-

chemical copolymerization of aniline with alkyl ring-

substituted anilines. Macromolecules 1990;23(3):758–64.

[36] Adams PN, Laughlin PJ, Monkman AP, Kenwright AM.

Low temperature synthesis of high molecular weight

polyaniline. Polymer 1996;37(15):3411–7.