Biosensors and Bioelectronics 28 (2011) 221–226 Contents lists available at ScienceDirect Biosensors and Bioelectronics j our na l ho me page: www.elsevier.com/locate/bios Real-time detection of 1 integrin expression on MG-63 cells using electrochemical impedance spectroscopy Chun-Yen Lin a,1 , Nai-Chia Teng a,1 , Sung-Chih Hsieh a , Yung-Sheng Lin b , Wei-Jen Chang a , Sheng-Yi Hsiao c , Hui-Shun Huang c , Haw-Ming Huang d,∗ a School of Dentistry, College of Oral Medicine, Taipei Medical University, 250, Wu-Hsing Street, Taipei, Taiwan b Department of Applied Cosmetology & Graduate Institute of Cosmetic Science, Hungkuang University, 34, Chung-Chie Road, Shalu, Taichung, Taiwan c Nano-Technology Division, Instrument Technology Research Center, National Applied Research Laboratories, 20, R&D Rd. VI, Hsinchu Science Park, Hsinchu, Taiwan d Graduate Institute of Biomedical Materials and Tissue Engineering, College of Oral Medicine, Taipei Medical University, 250, Wu-Hsing Street, Taipei, Taiwan a r t i c l e i n f o Article history: Received 11 May 2011 Received in revised form 11 July 2011 Accepted 11 July 2011 Available online 3 August 2011 Keywords: ITO Immunosensor Integrin MG-63 Electrochemical impedance spectroscopy a b s t r a c t Beta 1 integrin is a membrane protein responsible for attachment and migration of osteosarcoma cells. In this study, expression of 1 integrin on MG-63 cells, a human osteogenic sarcoma cell line, was mon- itored using electrochemical impedance spectroscopy (EIS). ITO-based biochips were developed using a semiconductor technique. Differences in electric resistance (R) were measured continuously when cells binding with anti-1 integrin antibody coagulated with nano-scale gold particles. The results of the EIS system were compared with traditional immunofluorescence staining. We found that sample chambers with higher cell densities had larger R values. When the cell densities increased from 5 × 10 4 cells/ml to 5 × 10 5 cells/ml, the R value dose-dependently increased from 14  to 37 . In addition, a highly linear relationship (correlation coefficient, 0.921) was found between the R values and the corresponding fluorescence intensities (p < 0.05). These results suggest that electrochemical impedance spectroscopy can be a useful tool for evaluating 1 integrin expression on cell membranes. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Electrochemical impedance spectroscopy (EIS) is a well- established technique for studying electrochemical systems for industrial applications. Since the early 1990s, EIS has been used to quantify bacterial content in solution. Using this technique, metabolic products in bacterial cultures act as electrolytes, and the resistance changes detected from the electrode can represent the activity of the microorganisms (Felice et al., 1999). Recently, EIS biosensors were developed for protein, oligonu- cleotide, and antibody detection by immobilizing one layer of a probe molecule on a conductive electrode (Suni, 2008). For exam- ple, various protein probes were coated on the conductive material surfaces. After binding to its specific antibody, the measured resis- tance changed due to the alternation of protein concentration (Berney et al., 1998; Kanungo et al., 2002). However, since the target cells are destroyed during the protein isolation stage, this method cannot be used for real-time and continuous detection of biomolecules. ∗ Corresponding author. Tel.: +886 2 2736 1661x5128; fax: +886 2 2736 2295. E-mail address: hhm@tmu.edu.tw (H.-M. Huang). 1 These authors contributed equally to this work. Electric cell-substrate impedance sensing (ECIS TM , Applied Bio- Physics, NY, USA), a commercialized EIS system developed by Giaever and Keese, allows impedance properties of attached and spreading cells to be measured in real time (Giaever and Keese, 1991, 1993). In the system, a 4000 Hz sinusoidal current (lower than 1 A) is continuously passed through the cells on small gold electrodes during the culture period, and the impedance of the cells is monitored by a lock-in amplifier. Although the ECIS TM system is able to detect changes in impedance caused by changes in cell proliferation (Guo et al., 2006; Xiao and Luong, 2003, 2005; Yeon and Park, 2005), morphology (DePaola et al., 2001), attachment and spreading (Wegener et al., 2000; Liu et al., 2007), motility (Chen et al., 2008; Jiang et al., 2009), and cytotoxicity (Opp et al., 2009; van der Schalie et al., 2006), few studies have reported on the use of ECIS TM to continuously detect changes in protein expression in living cells. Integrins are transmembrane glycoproteins composed of  and  subunits. The non-covalent combinations of the  and  subunits result in heterodimers with different structures, each providing a specific binding property to extracellular matrix proteins and cellu- lar ligands released from other cells. It is well known that integrins play an important role in various cellular functions, including pro- liferation, differentiation, adhesion, and migration on extracellular substrates (Chen et al., 2008; Cruet-Hennequart et al., 2003; Levy 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.07.022