Fabrication of a highly sensitive disposable immunosensor based on indium tin oxide substrates for cancer biomarker detection Mehmet Çetin Canbaz, Mustafa Kemal Sezgintürk ⇑ Division of Biochemistry, Department of Chemistry, Faculty of Science, Namık Kemal University, Tekirdag ˘, Turkey article info Article history: Received 7 August 2013 Received in revised form 7 October 2013 Accepted 9 October 2013 Available online 17 October 2013 Keywords: Cancer biomarkers Biosensor HER-3 ITO Single frequency impedance abstract Anti-HER-3 antibody was used for the first time in a disposable immunosensor based on indium tin oxide (ITO) substrate for HER-3 quantification. Anti-HER-3 was immobilized onto ITO substrate by 3-aminopro- pyl)triethoxysilane (APTES) and glutaraldehyde. This highly sensitive immunosensor was capable of detecting concentrations of HER-3 down to the femtogram/ml level by investigating changes in the charge transfer resistance (R ct ) using electrochemical impedance spectroscopy (EIS). Construction of ITO layers was carefully investigated using a broad range of techniques such as voltammetry, EIS, atomic force microscopy (AFM), and scanning electron microscopy (SEM). Meanwhile, in an immunosensor sys- tem, the ‘‘single frequency impedance’’ technique was first used for characterization of interaction between HER-3 and anti-HER-3. Eventually, the proposed ITO-based immunosensor was applied to arti- ficial serum samples spiked with HER-3. Ó 2013 Elsevier Inc. All rights reserved. HER-3 is a type of transmembrane growth factor receptor of the human epidermal growth factor receptor. It can activate intracellu- lar signaling pathways in response to extracellular signals [1,2]. HER-3 itself is an incomplete receptor functionally, and conse- quently it is a dependent protein [3]. Many studies have reported that tumor progression and reduced survival of patients with breast [4], ovarian [5], and pancreatic [6] cancers, gastric carci- noma [7], malignant melanoma and metastases [8], and head and neck squamous cell carcinoma were considerably associated with overexpression of HER-3. Moreover, HER-3 overexpression is importantly correlated with poor prognosis [9] and worse metasta- sis-free survival [10] in colorectal carcinomas. For instance, it has been revealed that both HER-3 messenger RNA and protein were upregulated in human breast cancers, such that in human breast cancers HER-3 overexpression has been reported in the ratio of 50–70% [11,4,12]. In addition, it seems to be associated with metastasis and local recurrence [13,14]. In this study, because of the vital importance of HER-3, an immunosensor based on indium tin oxide (ITO) 1 substrate as a working electrode was developed. Because of its good electrical con- ductivity, ITO is one of the most widely used transparent conducting oxides. Transparent ITO thin films on flexible substrates such as polyethylene terephthalate (PET) have many applications. It has been reported that they can be used in plastic liquid crystal display devices, transparent electromagnetic shielding materials, flexible electro-optical devices, heat reacting mirrors, and the like [15]. Various ITO-based immobilization techniques have led to new applications in the construction of biosensors. For example, gold nanoparticles were self-assembled onto an ITO electrode to prepare a modified sandwich-type electrochemical immunoassay platform for determination of vascular endothelial growth factor [16]. In an- other study, ITO was covered by a poly(dopamine) layer for biomol- ecule immobilization [17]. However, for more reliable and stable biosensor systems based on ITO substrates, more developments are needed to create useful immobilization strategies. Electrochemical impedance spectroscopy (EIS) has become an efficient method that is used for many chemical and physical pro- cesses. Hence, it is quite often applied in fabrication of immuno- sensors. In a typical EIS-based immunosensor, after any biorecognition event, charge transfer resistance (R ct ) usually in- creases, and this change in R ct can be used for detection of any sub- stances. However, in this study, a novel impedance method was applied to the immunosensor for the first time. ‘‘Single frequency impedance’’ was performed to reveal binding characteristics be- tween HER-3 and anti-HER-3. The focus of this study was on a simple immobilization proce- dure for anti-HER-3, development of a highly sensitive and dispos- able HER-3 biosensor, and its impedimetric characterization. The current study is the first to use anti-HER-3 as a bioreceptor in an immunosensor system for HER-3 analysis. We also identified 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.10.014 ⇑ Corresponding author. Fax: +90 282 250 99 25. E-mail addresses: msezginturk@nku.edu.tr, msezginturk@hotmail.com (M.K. Sezgintürk). 1 Abbreviations used: ITO, indium tin oxide; PET, polyethylene terephthalate; EIS, electrochemical impedance spectroscopy; SEM, scanning electron microscopy; BSA, bovine serum albumin; APTES, 3-aminopropyltriethoxysilane; CV, cyclic voltamme- try; AFM, atomic force microscopy; SAM, self-assembled monolayer. Analytical Biochemistry 446 (2014) 9–18 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio