This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

Author's personal copy

Immobilization of cholesterol oxidase onto electrochemically polymerized film ofbiocompatible polyaniline-Triton X-100

Raju Khan a,⁎, Ajeet Kaushik a, A.P. Mishra b

a Biomolecular Electronics and Conducting Polymers Research Group, National Physical Laboratory, New Delhi 110012, Indiab Department of Science and Technology, Ministry of Science and Technology, Technology Bhawan New Delhi-110016, India

a b s t r a c ta r t i c l e i n f o

Article history:Received 12 May 2008Received in revised form 20 October 2008Accepted 3 November 2008Available online 12 November 2008

Keywords:BiosensorPANI-TX-100ImmobilizationCholesterol oxidase

Cholesterol oxidase (ChOx) has been successfully immobilized on electrochemically-polymerized polyaniline(PANI) with Triton X-100 (TX-100) thin film through covalent bonding. The change in the surface morphologyof PANI-TX-100 thin film due to immobilization of ChOx was investigated using Atomic Forced Microscopy.The absorption band at 660 nm in the UV–vis absorption spectra and the presence of the infrared markerscorresponds to the amide group in ChOx/PANI-TX-100 film confirm the immobilization of ChOx in PANI-TX-100 matrix. The broad hump in cyclic voltammogram and an increase in charge transfer resistance inelectrochemical impedance measurement of ChOx/PANI-TX-100/ITO revealing covalently immobilization ofChOx on PANI-TX-100 networks.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Cholesterol and its fatty acid esters are important compounds forhuman beings because they are the components of nerve and braincells and are precursors of other biological materials such as bile acidand steroid hormones [1]. The estimation of cholesterol in blood is animportant parameter for the diagnosis and prevention of several heartdiseases, hypertension, cerebral thrombosis, and arteriosclerosis witha strong positive correlation between total cholesterol in human bloodserum and heart diseases [2–4]. Various analytical methods have beenused for cholesterol assays including colorimetric, spectrometric andelectrochemical methods [5,6]. Several cholesterol biosensors includ-ing enzymatic and non-enzymatic methods have been reported [7–11]. However, these often have certain difficulties like lack ofspecificity and selectivity due to the presence of various interferingreactions and the use of unstable and corrosive reagents.

Cholesterol sensor is multi enzymatic electrode system in whichhorseradish peroxidase and cholesterol oxidase (ChOx) is simultaneouslyimmobilized within a polymeric film on the surface of pyrolitic graphiteelectrode [12,13]. Electrochemical detection systems for cholesterol assayare frequently based on monitoring the consumption of oxygen or therate of production of hydrogen peroxide by an enzymatic reaction.

Conducting polymers such as polyaniline (PANI), polythiophene,polyphenylene and polypyrrole have been utilized for the developmentof biosensors for application in clinical diagnostics. Among these, PANI

exhibits unique advantage for the detection of various clinicallyimportant metabolites i.e., glucose, lactate, urea, galactose and choles-terol etc. However, the difficult processability and poor thermal stabilityhas to overcome for the successful application PANI in the field ofbiosensor. In order to overcome this obstacle the synthesis of PANI withsurfactants may be an attractive alternate. Surfactants effect the locuspolymerization by using the emulsion or inverse emulsion pathways,and thus modify the supermolecular structure and surface morphologyof PANI [14–20]. Furthermore, surfactant improves the properties PANIsuch as conductivity, stability and solubility [19] in organic solvents,which makes PANI as promising polymer matrices for the bio sensingapplication. This is always a matter of great interest to develop betterPANI based polymeric matrices for the immobilization of cholesteroloxidase and helpful in the development of cholesterol biosensors.

In this study, we successfully immobilized ChOx on the surface ofelectrochemical polymerized thin film of PANIwith nonionic surfactant,Triton X-100 [4-(1,1,3,3-tetramethylbutyl) phenyl polyethylene glycoli.e., TX-100]. The presence of TX-100 in PANI network affects the surfacemorphology along with the electrical conductivity. The immobilizationof ChOx increases the charge transfer resistance of the sensing electrodedue to the covalent bonding suggest its potential application forcholesterol biosensor.

2. Experiment

2.1. Chemicals and reagents

Hydrochloric acid (32%) and anilinemonomer (99.5%) of analyticalgrade were purchased from Fluka, India. Nonionic surfactants i.e.,

Materials Science and Engineering C 29 (2009) 1399–1403

⁎ Corresponding author. Analytical Chemistry Division, North-East Institute of Science &Technology, Jorhat-785006, Assam, India. Tel.: +91 376 2370012; fax: +91 376 230011.

E-mail address: khan.raju@gmail.com (R. Khan).

0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.msec.2008.11.001

Contents lists available at ScienceDirect

Materials Science and Engineering C

j ourna l homepage: www.e lsev ie r.com/ locate /msec

Author's personal copy

TritonX-100 (99%) was purchased from Sigma Aldrich, USA. Electro-chemical polymerization and cyclic voltammetric measurementswere carried out on a Potentiostate/Galvanostat (Princeton AppliedResearch model).

2.2. Electropolymerization of PANI thin film with TX-100

Aniline monomer were double distilled and kept under reducedpressure before polymerization. The electrochemical polymeriza-tion of PANI films with TX-100 was carried outrange in thepotential from −200 to +900 mV using chronoamperometrymethod on indium–tin-oxide (ITO) substrate with three-electrodecell configuration. Platinum foil was used as a counter electrode,Ag/AgCl as a reference electrode and an ITO glass plate as theworking electrode. The electrochemical polymerization solutionconsisted of aniline and TX-100 (1:1) with 1 M HCl, used assupporting electrolyte. The polymer thin film was rinsed manytimes by the deionized water. The proposed mechanism for theelectrochemical polymerization of PANI with TX-100 is shown inScheme 1.

Scheme 1. Proposed mechanism for the electrochemically polymerization of polyaniline thin film with TX-100.

Scheme 2. Proposed mechanism for Covalently immobilization of ChOx on PANI-TX-100 polymer matrix.

Fig. 1. UV–vis spectra of electrochemically polymerized thin film of (a) PANI-TX100 and(b) PANI-TX-100/ChOx bioelectrode.

1400 R. Khan et al. / Materials Science and Engineering C 29 (2009) 1399–1403

Author's personal copy

2.3. Immobilization of PANI-TX-100 thin film

Freshly prepared solution of cholesterol oxidase was prepared in10% TX-100 and stored at 4 °C. The surface of PANI-TX-100 thin filmwas immobilized with ChOx by covalent linkage through glutaralde-hyde (0.1%) using well-known chemistry. ChOx solution (10 μL) wasspread on the PANI-TX-100 film (containing covalently linkedglutaraldehyde) for the immobilization and subsequently kept over-night to allow the uniform distribution of enzymes on the surface ofPANI-TX-100 matrix. The presences of any unbounded or looselybound enzyme molecules were removed after rinsing PANI-TX-100/ChOx thin film with deionized water.

The surface morphology of thin filmwas studied using and AtomicForce Microscopy (AFM). The structural properties of electrochemi-cally polymerized PANI-TX-100 and PANI-TX-100/ChOx thin film havebeen studied using Fourier Transform Infrared spectrophoto-meter (Perkin Elmer) in the frequency range from 400–4000 cm−1.UV–visible spectrophotometer (Ocean Optics) in the range from 300to 1000 nm is used to study the optical properties of depositedpolymer matrices. The electrical properties of thin film in PANI-TX-100/ITO and ChOx/PANI-TX-100 configuration were measured usingelectrical impedance spectroscopy.

3. Results and discussion

3.1. Proposed mechanism of covalent immobilization of ChOx on PANI-TX-100 matrix

Glutaraldehyde is a bifunctional compound mainly used as crosslinker for the chemical modifications of proteins and polymers. Thisbifunctional compound links covalently to the amine groups of lysineor hydroxylysine in the protein molecules (ChOx) creating a structuremore stable than that attained by the physical aggregation. This typeof bonding reinforces the compact tertiary structures resulting inprotein stabilization against pH inactivation for immobilizing therespective enzymes. In the electrochemical polymerization of PANI-TX-100, large number of free NH2 groups available at the terminal ofPANI unit, which bind with the NH2 groups of ChOx with glutar-aldehyde as a cross-linker. Whereas, the CfO group of glutaraldehydebinds with the NH2 group via condensation reaction with theelimination of water molecule resulting in covalent linkage of ChOxwith PANI. The tentative mechanism of the immobilization of ChOx onPANI-TX-100 film is shown in Scheme 2.

3.2. Optical properties

The UV–visible absorption spectra of electrochemically polymer-ized PANI-TX-100 and ChOx-PANI-TX-100 films are shown in Fig. 1.

Fig. 2. FTIR spectra of electrochemically polymerized thin film of (a) PANI-TX100 and(b) PANI-TX-100/ChOx bioelectrode.

Fig. 3. AFM images of (a) PANI-TX-100 film and (b) PANI-TX-100/ChOx bioelectrode.

1401R. Khan et al. / Materials Science and Engineering C 29 (2009) 1399–1403

Author's personal copy

The broad absorption band in the range from 300 nm to 450 nm isobserved in the spectra of PANI-TX-100 film (curve a). In the pure PANIthe absorption band at 320 nm is attributed to the л–л⁎ transitionwithin benzenoid segment and at 420 nm is related to the polaronband–л⁎ transition. Both bands are merged in PANI matrix with TX-100 due to overlapping of absorption band corresponds to the л–л⁎electronic transition in both PANI and TX-100 (curve a).

The absorption band at 850 nm is assigned to the л-localisedpolaron bands of doped PANI in emaraldine salt form. It may be notedfrom Fig. 1 that the absorption band in the range 300 to 450 nm isshifted to the higher wavelength region after immobilizing of ChOx onthe surface PANI-TX-100 thin film and may be attributed to theoverlapping of л–л⁎ transition and polaron band–л⁎ transition ofPANI-TX-100 with ChOx. After ChOx immobilization via glutaralde-hyde chemistry, the absorption band at 850 nm shift to lowerwavelength at 660 nm (curve b). This reveals that due to glutar-alhehyde the protonated state (–N+H–) of PANI deprotonated to NH2

i.e., emaraldine base form. It is also noticed that insulatingcharacteristic of glutaraldehyde decreases in the conjugation in thephenyl–phenyl ring due to steric hindrance resulting in decrease bandpeak position in the absorbance spectra of ChOx/PANI-TX-100/ITO.The observed shift in the absorption bands (curve b) confirms theimmobilization of ChOx on the surface of PANI-TX-100 thin film mayenhance the sensing properties [21].

FTIR spectrum of electrochemically polymerized thin films ofPANI-TX-100 and ChOx/PANI-TX-100 is shown in Fig. 2. The infraredspectrum of PANI-TX-100 consist the entire absorption bands of TX-100 at 1600 cm−1, 1460 cm−1, 1351 cm−1, 1298 cm−1, 1246 cm−1,1184 cm−1, 1124 cm−1 and 953 cm−1 [22]. The absorption bandsobserved at 1570 and 1469 cm−1 in PANI-TX-100 assigned to the non-symmetric vibration mode of CfC in quinoid and benzenoid ringsystem in polyaniline respectively. The C–N stretching vibration modein aromatic amine nitrogen (quinoid system) in doped polyaniline isfound at 1297 cm−1, corresponding to the oxidation or protonationstate. The absorbance peak at 1237 cm−1 is attributed to C–Nstretching vibrationmode in benzenoid ring system of polyaniline dueto the conducted protonated form. The in-plane vibration of C–Hbendingmode in NfQfN, Q–N+H–B or B–N+H–B (where Q=quinoidand B= benzenoid) is observed at 1156 cm−1 in the FTIR spectra. Thepresence of this absorption band is expected due to the polymeriza-tion of PANI i.e., polar structure of the conducted protonated form. Theabsorption bands at 884 and 816 cm−1 (curve a) are attributed to thearomatic ring and out of plane C–H deformation vibrations for 1,4-disubstituted aromatic ring system.

Infrared spectra of ChOx-PANI-TX-100 consists of all the absorptionband of PANI-TX-100 along with the characteristics bands of ChOx at3257 cm−1, 1720, cm−1 1663 cm−1 and 1511 cm−1 (curve b). Thesemodes are assigned to the amide bonds [23] confirms the immobiliza-tion of ChOx on PANI-TX-100 thin film. It may be noted that the modeat 3251 cm−1 becomes broad in the ChOx/PANI-TX-100 structure andmay be due to the covalent bonding of ChOx.

3.3. Surface morphology

The surfacemorphology of electrochemically deposited thin film ofPANI-TX-100 was investigated using AFM and shown in Fig. 3. A roughmicrostructure was observed in the AFM image of PANI-TX-100polymermatrix (image a) with the average roughness of about 60 nm.This might be due the interfacial polymerization of aniline in thevicinity of the micelle–water interface that produces nano-porouspolymer growth that further supported by Scheme 1 [24]. The porousmorphology of PANI-TX-100 polymer changes into another regularwith increased roughness (80 nm, image b). This may be due touniform distribution of ChOx onto PANI-TX-100 matrix confirms theimmobilization of ChOx on PANI-TX-100 polymeric matrix. It may benoted that the affinity of ChOx is very strong with TX-100 and its

incorporation in PANI matrices may prevent the leakage of enzymeduring the biosensing. The rough surface morphology has addedadvantage and enzyme are expected to adsorb strongly on the surfaceof PANI-TX-100. The observed change in the morphology of PANI bythe incorporation of TX-100 may provide effective confirmation forthe immobilization of ChOx.

3.4. Cyclic voltammogram analysis

The cyclic voltammeter of experiment was carried out in 0.1 Mphosphate buffer (50 saline mM, pH 7.0), PANI-TX-100/ITO and ChOx-PANI-TX-100/ITO working electrode, Ag/AgCl reference electrode andPt wire as a counter electrode. The PANI-TX-100/ITO and ChOx/PANI-TX-100/ITO thin film electrode structure at scan rate of 50 mV/s areshown in Fig. 4. In PANI-HCl, the redox couple occurred at approxi-mately +200 mV is assigned to the transition from the reducedleucoemeraldine (LE) state to partially oxidized emeraldine state (EM)and the redox couple at+800 mV corresponding to the transition fromLE to pernigraniline (PE) state, and is accompanied by the oxidation ofaniline monomer [25]. A less intense redox couple is observed at+500 mV is generally assigned to the redox reaction of p-benzoqui-none [26]. The redox peak potentials of PANI-TX-100 have been shiftedappreciably with respect to PANI-HCl. The observed shift may be dueto electrostatic interactions between the head group of TX-100 andcharged aniline at the hydrophilic interface of the micelles. The ChOx/

Fig. 4. Cyclic voltammogram of (a) PANI-TX-100 and (b) PANI-TX-100/ChOx in(phosphate buffer (50 mM, pH 7.0, 0.9% NaCl) at scan rate 50 mV s−1.)

1402 R. Khan et al. / Materials Science and Engineering C 29 (2009) 1399–1403

Author's personal copy

PANI-TX-100/ITO electrode structure shows a broad hump (curve b)in the comparison of PANI-TX-100/ITO electrode suggesting that theimmobilization of ChOx slow down the redox process during theelectrochemical reaction.

3.5. Electrochemical impedance measurements

Electrochemical impedance spectrograph of PANI-TX-100/ITO andChOx/PANI-TX-100/ITO thin film electrode was measured in phos-phate buffer (50 mM, pH 7.0, 0.9% NaCl) containing [Fe(CN)6]−3/−4

(5 mM) in the frequency range from 0.01 to 105 Hz using the Autolabpotentiostat/galvanostat (Eco Chemie, The Netherlands). The Nyquistplot for PANI-TX100/ITO electrode shows (Fig. 5) semicircle, with a RCTvalue of 1.301×103 Ω (curve a). The semicircle at higher frequenciescorresponding to the electron-transfer limited process and the linearportion at the lower frequencies that may be ascribed to the diffusionprocesses. The RCT value of ChOx/PANI-TX100/ITO bioelectrode isfound to be 2.03×102 Ω (curve b) that is lower than PANI-TX100/ITOelectrode. The decrease in RCT value may be attributed to the presenceof a large number of polarizable groups such as imine and amine etc.in the enzyme (ChOx) molecules onto the surface that get polarized atopen circuit potential. Polarization resulting in positive charge on thesurface enhances the mobility of redox probe [Fe(CN)6]3−/4− to theelectrode leading to the decrease of the charge transfer resistance.This decrease in RCT value indicates the binding of ChOx to PANI-TX100 matrix.

The observed result indicate the formation of a stable polymermatrix of PANI-TX-100. PANI-TX-100 thin film deposited on ITOconducting substrate was found to be strongly adherent and stable.Since TX-100 is generally used to dissolve ChOx enzyme for variousapplications because cholesterol is a springy soluble substance inaqueous solution and TX-100 play an important role in stabilizingactivity of ChOx [27]. The incorporation of TX-100 in the polymer

matrix in the present study and subsequently successful immobiliza-tion of ChOx through covalent bonding on the rough surface of PANI-TX-100may prevent the leakage of enzyme during the biosensing. Thework of its application for the cholesterol sensing is under process.

4. Conclusion

Cholesterol oxidase has been successfully covalently immobilizedon the conducting surface of polyaniline with TX-100 (PANI-TX-100)thin film. The presence of the absorption band at 660 nm in UV–visspectra and infrared markers at 3251, 1720, 1663 and 1551 cm−1

corresponding to amide group confirm the immobilization of ChOx onPANI-TX-100 polymer matrix. This is also confirmed by the surfacemorphology of immobilized ChOx/PANI-TX-100 electrode structure. Abroad hump in the cyclic voltammetric measurement and anincreased charge transfer resistance value of ChOx/PANI-TX-100/ITOelectrode confirm the covalently immobilization of ChOx on PANI-TX-100 polymer matrices.

Acknowledgement

Dr. Raju Khan thanks to Department of Science & Technology (DST)for financial support DST-Fast Track Young Scientist sponsoredprojects SR/FTP/CS-77/2007 is gratefully acknowledged. We thank toDr. Vikram Kumar (Director, NPL) and BECPRL group for infrastructuresupport.

References

[1] S. Singh, P.R. Solanki, M.K. Pandey, B.D. Malhotra, Anal. Chim. Acta 568 (2006) 126.[2] B.D. Malhotra, A. Chaubey, Sens. Actuators B, Chem. 91 (2003) 117.[3] S. Singh, A. Chaubey, B.D. Malhotra, Anal. Chim. Acta 502 (2004) 229.[4] V. Shumyantseva, G. Deluca, T. Bulko, S. Carrara, C. Nicolini, S.A. Usanov, A.

Archakov, Biosens. Bioelectron. 19 (2004) 971.[5] B. Zak, R.C. Dickenmann, Am. J. Clin. Pathol. 24 (1954) 1307.[6] B. Zak, Am. J. Clin Pathol. 27 (1957) 583.[7] I. Karube, K. Hara, H. Matsuoka, S. Suzuki, Anal. Chim. Acta 139 (1982) 127.[8] J.C. Vidal, J. Espuelas, E. Garcia-Ruiz, Anal Lett. 35 (2002) 837.[9] S. Brahim, D. Narinesingh, A. Guiseppi-Elie, Anal. Chim. Acta 448 (2001) 27.[10] C. Bongivanni, T. Ferri, A. Poscia, M. Varalli, R. Santucci, A. Desideri, Bioelec-

trochemistry 54 (2001) 17.[11] M.A.T. Gilmartin, J.P. Hart, Analyst 119 (1994) 2331.[12] T. Ferri, A. Poscia, R. Santucci, Bioelectrochem. Bioenerg. 44 (1998) 177.[13] T. Ferri, A. Poscia, R. Santucci, Bioelectrochem. Bioenerg. 45 (1998) 221.[14] S.P. Armes, M. Aldissi, J. Chem. Soc. Chem. Commun. (1989) 88.[15] N. Kohut-Svelko, S. Reynaud, J. Francois, Synth. Met. 150 (2005) 107.[16] P.J. Kinlen, J. Liu, Y. Ding, C.R. Graham, E.E. Remsen, Macromolecules 31 (1998)

1735.[17] H. Xia, Q. Wang, J. Nanopart. Res. 3 (2001) 40.[18] P.S. Rao, S. Subrahmanya, D.N. Sathyanarayana, Synth. Met. 128 (2002) 311.[19] P.S. Rao, D.N. Sathyanarayana, S. Palaniappan, Macromolecules 35 (2002) 4988.[20] A.D.W. Carswell, E.A. O'Rear, B.P. Grady, J. Am. Chem. Soc. 125 (2003) 14793.[21] M. Jayakannan, P. Anilkumar, A. Sanju, Eur. Polym. J. 42 (2006) 2623.[22] P. Kluson, P. Kacer, T. Cajthaml, M. Kalaji, J. Mater. Chem. 11 (2001) 644.[23] S.K. Arya, A.K. Prusty, S.P. Singh, P.R. Solanki, M.K. Pandey, M. Datta, B.D. Malhotra,

Anal. Biochem. 363 (2007) 210.[24] X. Zhang, S.K. Manohar, Chem. Commun. 20 (2004) 2360.[25] K.R. Prasad, N. Munichandraiah, Synth. Met. 123 (2001) 459.[26] J.C. Cooper, E.A.H. Hall, Biosens. Bioelectron. 7 (1992) 473.[27] H. Wang, S. Mu, Sens. Actuators B 56 (1999) 22.

Fig. 5. Electrochemical impedance spectra of (a) PANI-TX-100 film and (b) PANI-TX-100/ChOx bioelectrode (50 mM, pH 7.0, 0.9% NaCl) at scan rate 50 mV s−1.)

1403R. Khan et al. / Materials Science and Engineering C 29 (2009) 1399–1403