Label-free electrochemical IgE aptasensor based on covalent attachment of aptamer onto multiwalled carbon nanotubes/ionic liquid/chitosan nanocomposite modified electrode Somayeh Khezrian a , Abdollah Salimi a,b,n , Hazhir Teymourian a , Rahman Hallaj a a Department of Chemistry, University of Kurdistan, P. O. Box 416, Sanandaj, Iran b Research Center for Nanotechnology, University of Kurdistan, P.O. Box 416, Sanandaj, Iran article info Article history: Received 14 June 2012 Received in revised form 25 November 2012 Accepted 4 December 2012 Available online 20 December 2012 Keywords: Label-free aptasensor Immunoglobulin E Chitosan Ionic liquid Carbon nanotubes abstract A simple, sensitive and label-free aptamer-based biosensor for the detection of human immunoglobulin E (IgE) is developed using the electrochemical transduction method. A special immobilization interface consisting of multiwalled carbon nanotubes/ionic liquid/chitosan nanocomposite (MWCNTs/IL/Chit) is utilized to improve the conductivity and performance characteristics of the biosensor as well as to increase the loading amount of aptamer DNA sequence. A 5’-amino-terminated aptamer is covalently attached onto MWCNTs/IL/Chit modified glassy carbon (GC) electrode via a linker of glutaraldehyde (GA). Methylene blue (MB) is used as an electrochemical indicator which is intercalated into the aptamer through the specific interaction with its guanine bases. In the absence of IgE, MB bound to the aptamer produces a strong differential pulse voltammetric (DPV) signal. But when IgE exists, the intercalated MB releases from the aptamer, resulting an obviously decreased DPV signal. This phenomenon can be applied for human IgE detection. The peak current of MB linearly decreases with the concentration of IgE over a range of 0.5–30 nM with a detection limit of 37 pM. By using Bovine serum albumin (BSA) and lysozyme, the excellent specificity of this sensing system for the detection of IgE is also demonstrated. Finally, the proposed aptasensor is successfully used to IgE analysis in human serum sample. The obtained result is well agreed with the value obtained by the standard ELISA method. The herein described approach is expected to promote the exploitation of aptamer-based biosensors for protein assays in biochemical and biomedical studies. & 2012 Elsevier B.V. All rights reserved. 1. Introduction Since the pioneering studies on aptamers (Ellington and Szostak, 1990; Tuerk and Gold, 1990), substantial research efforts have been directed in order to apply the aptamers as the active sensing materials in different detection schemes (Zayats et al., 2006), led to the development of various electrical (Radi et al., 2005; Xu et al., 2005; Wu et al., 2009), microgravimetric (Pavlov et al., 2004; Yao et al., 2009) and optical aptasensors (Nutiu and Li, 2003; Ho and Leclerc, 2004; Gokulrangan et al., 2005). Aptamers are artificial nucleic acid ligands showing specific binding affinity for amino acids, drugs, proteins and other small molecules, which can be screened through the systematic evolu- tion of ligands by exponential enrichment (SELEX) process from random RNA or DNA libraries (Tuerk and Gold, 1990; Ellington and Szostak, 1990). Compared with antibodies, aptamers possess some outstanding features, including high specificity of binding affinity, nice stabilization, easy synthesis and modification with electrochemical active markers, optical dyes, enzymes and other desired substances. Furthermore, aptamers can reversibly capture and release their target molecule. It is more facile for the aptamer to transduce the recognition events into detectable signals (Kang et al., 2008). Among the various sensing devices developed so far, the detection methods of label-free, aptamer-based electrochemical biosensors have received an increasing amount of attention due to their inherent advantages such as simplicity, high sensitivity, low cost and high stability (Cheng et al., 2007; Feng et al., 2008; Wang et al., 2009a; Yan et al., 2011). Many of these electrochemical methods are based on electrochemical redox indicators that bind to the nucleic acids via electrostatic interactions or intercalation. Methylene blue (MB) is a widely-used redox indicator that belongs to phenothiazine family and is an aromatic cationic dye showing optically and electrochemically active properties. It has been proved that MB could intercalate the nucleic acid duplexes Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics 0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.12.006 n Corresponding author at: Department of Chemistry, University of Kurdistan, P.O. Box 416, Sanandaj, Iran. Tel.: þ98 871 6624001; fax: þ98 871 6624008. E-mail addresses: absalimi@uok.ac.ir, absalimi@yahoo.com (A. Salimi). Biosensors and Bioelectronics 43 (2013) 218–225