Sensors and Actuators B 141 (2009) 518–525 Contents lists available at ScienceDirect Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb Direct electrochemistry of cytochrome c and biosensing for hydrogen peroxide on polyaniline grafted multi-walled carbon nanotube electrode Kwang-Pill Lee a,b , Anantha Iyengar Gopalan a,b,∗ , Shanmugasundaram Komathi a a Department of Chemistry Graduate School, Kyungpook National University, Daegu 702-701, South Korea b Nano Practical Application Center, Daegu 704-801, South Korea article info Article history: Received 31 March 2009 Received in revised form 27 May 2009 Accepted 18 June 2009 Available online 27 June 2009 Keywords: Multi-walled carbon nanotube Grafted polyaniline Cytochrome c Direct electron transfer Hydrogen peroxide abstract Cytochrome c (cyt c) was immobilized into a matrix consisting of polyaniline (PANI) and multi-walled carbon nanotubes (MWNT) by a new strategy. First, PANI chains were grafted onto MWNT through electropolymerization. Second, the amine groups in PANI chains were oxidized at an applied potential of +0.80V to acquire positive charges that would effectively immobilize negatively charged cyt C. The ITO/MWNT-g-PANI(O)/cyt c electrode exhibited a pair of redox peaks with a peak potential separation (anodic to cathodic) of 0.25V (vs Ag/AgCl) in 0.1M phosphate buffer (pH 7.0). The results demonstrated that ITO/MWNT-g-PANI(O)/cyt c promoted direct electron transfer between cyt c and electrode with a high electron transfer rate constant (17 s -1 ). The ITO/MWNT-g-PANI(O)/cyt c electrode catalyzes the reduction of H 2 O 2 . The ITO/MWNT-g-PANI(O)/cyt c biosensor displays an amperometric response to H 2 O 2 with a linear concentration range from 0.5 M to 1.5 mM (r = 0.99, n = 12), a high sensitivity (32.2 Am M -1 ) and fast response (9 s) and detection limit of 0.3 M (S/N = 3). © 2009 Elsevier B.V. All rights reserved. 1. Introduction Cytochrome c (Cyt c), a heme containing metalloprotein, attracts research interest in bioelectrochemistry due to its important role in respiratory chain in mitochondria [1]. Cyt c exists in the cytosol between the inner and outer membranes of mitochondria. Studies on the oxidation–reduction reactions and kinetics of the electron transfer processes of cyt c are of great significance due to their importance in the fabrication of biosensors [2]. Cyt c exhibits only a weak electrochemical response at a bare electrode due to its extremely slow electron transfer kinetics at electrode/solution interface [3]. The surface of the bare electrode is blocked for the electron transfer by the irreversible adsorption of cyt c [4]. Modification of the electrode surface is required to enhance the electron transfer rate of cyt c. Utilizing the direct electron transfer (DET) between cyt c and underlying electrode, biosensors, enzymatic bioreactors and biomedical devices have been fabricated [5,6]. Many developments have been taken place towards the fab- rication and electrochemical characteristics of modified electrodes [7,8] which indirectly contribute to the development of ampero- ∗ Corresponding author at: Department of Chemistry Graduate School, Kyung- pook National University, 1370, Sankyuk Buk-gu, Daegu 702-701, South Korea. Tel.: +82 53 950 5901; fax: +82 53 952 8104. E-mail addresses: kplee@knu.ac.kr (K.-P. Lee), algopal99@gmail.com (A.I. Gopalan). metric biosensors [9–11]. One of the important problems in the fabrication of modified electrode with cyt c is the difficulty for the control of thickness and uniformity of the modifying layer. As a result, the reproducibility and the performance of the modified electrodes with cyt c are affected. A layer or film of carbon nan- otubes (CNTs) or a conducting polymer (CP) would function as a suitable matrix for the electrode to immobilize cyt c. CNTs show great potentials for the fabrication of highly sensi- tive biosensors [12–14]. Cyt c immobilized CNTs based modified electrodes have shown improved sensitivity and selectivity towards analytes [10]. Multi-walled carbon nanotubes (MWNT) were immo- bilized with cyt c and used as electrode to elucidate the electron transfer mechanism and electrocatalytic activity of H 2 O 2 [15]. The restricted solubility of CNTs necessitated the use of functionalized (non-covalent or covalent) CNTs or composites of CNTs for applica- tions. A biosensor has been fabricated by immobilizing an enzyme into the composites of CNTs [16]. Direct electron transfer (DET) from cyt c has been reported to occur at few of the CNT based modified electrodes [2,17–19]. The surface of CNTs is hydrophobic and limits the amount of enzyme immobilization. The hydrophobic surface of CNTs could be modified by functionalization. However, studies on the utilities of functionalized CNTs for immobilization of cyt c are scarce. CPs have emerged as promising materials for the immobiliza- tion of enzymes or proteins [20,21]. Composites consisting of a CP and CNT have exhibited properties of the individual compo- nents with synergistic effects [22,23]. CNTs/polymer composites 0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2009.06.039