Visualization of the membrane engineering concept: evidence for the specific orientation of electroinserted antibodies and selective binding of target analytes Anna Kokla a , Petros Blouchos a , Evangelia Livaniou b , Christos Zikos b , Sotiris E. Kakabakos b , Panagiota S. Petrou b and Spyridon Kintzios a * Membrane engineering is a generic methodology for increasing the selectivity of a cell biosensor against a target molecule, by electroinserting target-specific receptor-like molecules on the cell surface. Previous studies have elucidated the biochemical aspects of the interaction between various analytes (including viruses) and their homologous membrane-engineered cells. In the present study, purified anti-biotin antibodies from a rabbit antiserum along with in-house prepared biotinylated bovine serum albumin (BSA) were used as a model antibody-antigen pair of molecules for facilitating membrane engineering experiments. It was proven, with the aid of fluorescence microscopy, that (i) membrane-engineered cells incorporated the specific antibodies in the correct orientation and that (ii) the inserted antibodies are selectively interacting with the homologous target molecules. This is the first time the actual working concept of membrane engineering has been visualized, thus providing a final proof of the concept behind this innovative process. In addition, the fluorescence microscopy measurements were highly correlated with bioelectric measurements done with the aid of a bioelectric recognition assay. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: bioelectric recognition assay; biotinylated BSA; cell biosensors; cell membrane engineering; electroinsertion; membrane potential INTRODUCTION Membrane engineering is a generic methodology of artificially inserting (usually by electroinsertion) tens of thousands of recep- tor-like molecules on the cell surface, thus rendering the cell a selective responder against analytes binding to the inserted recep- tors. Receptor molecules can vary from antibodies to enzymes. The working assumption of the method is that attachment of the tar- get molecule to its respective receptor causes a change in the cell membrane structure, which is measurable as a change in the cell membrane potential. This technology is an alternative method to cell transfection for increasing cell specificity, a requirement dic- tated by the usually poor selectivity of cell-based assay and sensor systems, because of the uniform response of cells against a vastly large number of different molecules. Membrane-engineered cells have been developed and used as biorecognition elements in a plethora of applications, including the detection of superoxide (Moschopoulou and Kintzios, 2006; Moschopoulou et al., 2007, 2012), human viruses (Hepatitis B) (Perdikaris et al., 2009), plant viruses (Gramberg et al., 2012; Moschopoulou et al., 2008; Perdikaris et al., 2011), organic compounds such as 2,4,6-trichloroanisole (Varelas et al., 2010), aflatoxins (Larou et al., 2012), metabolic diseases, prions, foot-and-mouth disease virus, and blue- tongue virus (Kintzios, 2008). It has been previously demonstrated that the specific interac- tion between membrane-engineered cells and homologous target analytes is associated with changes in intracellular Ca 2+ traffic, which in turn affect cell membrane potential, as revealed by the selective inhibition of calcium ion pumps (Moschopoulou et al., 2012). In the same study, it was shown that cells, which were membrane-engineered with enzymes (superoxide dismutase [SOD]) as receptor-like molecules, acted as fully functional catalytic units, whereas the electroinserted SOD molecules retained their characteristic properties, as demonstrated by selective inhibition assays. However, similar studies have not been conducted with an- tibodies, which are more frequently used as recognition elements in membrane engineering experiments. In the present study, purified anti-biotin antibodies from a rabbit antiserum, along with in-house prepared biotinylated bovine * Correspondence to: S. Kintzios, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece. E-mail: skin@aua.gr a A. Kokla, P. Blouchos, S. Kintzios Department of Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece b E. Livaniou, C. Zikos, S. E. Kakabakos, P. S. Petrou Institute of Nuclear and Radiological Sciences & Technology, Energy and Safety, NCSR Demokritos, 153 10 Athens, Greece Abbreviations: BERA, bioelectric recognition assay.; PBS, phosphate buffer saline. Research Article Received: 24 May 2013, Revised: 17 July 2013, Accepted: 31 July 2013, Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jmr.2304 J. Mol. Recognit. 2013; 26: 627–632 Copyright © 2013 John Wiley & Sons, Ltd. 627