Research paper Detection of two different influenza A viruses using a nitrocellulose membrane and a magnetic biosensor Hyo-Bong Hong a, ⁎, Hans-Joachim Krause b , Ki-Bong Song a , Chel-Jong Choi c , Myung-Ae Chung a , Sung-won Son a , Andreas Offenhäusser b a IT Convergence Service Core Research Team, Electronics and Telecommunications Research Institute, 138 Gajeongno, Yuseoung-Gu, Daejeon, Republic of Korea b Institute of Bio and Nano Systems, IBN-2, Forschungszentrum Jülich, 52425 Jülich, Germany c School of Semiconductor and Chemical Engineering, Semiconductor Physics Research Center (SPRC), Chonbuk National University, Jeonju 561-756, Republic of Korea article info abstract Article history: Received 28 June 2010 Received in revised form 7 December 2010 Accepted 13 December 2010 Available online 21 December 2010 Here we describe a new analytical method for the detection of two influenza A viruses by nitrocellulose membrane and magnetic sensors that employ a special frequency mixing technique. The combination of the nitrocellulose membrane and magnetic bead detection permits a rapid assay procedure and excludes two steps (the development of color and the stop reaction) required for usual immunochemical detection methods such as ELISA. Quantitative virus detection was performed using magnetic beads conjugated with secondary antibody. The results were compared with conventional assay methods and with a dot-blot assay with fluorescence compound (FITC). Under optimum conditions, our new assay procedure is capable of detecting picograms of virus per well. This new method combining the nitrocellulose membrane and magnetic bead detection reduces analytical time and allows stable and repeatable analyses of samples in point-of-care applications. © 2010 Elsevier B.V. All rights reserved. Keywords: Influenza A virus Immunodetection Immunoflow assay Magnetic assay 1. Introduction Influenza A virus belongs to the RNA virus family Orthomyxoviridae that infects mammals, including human beings. It is generally transmitted through the air by coughs, sneezes, and direct contact with the contaminated materials such as body fluids (Subbarao and Katz, 2000). In order to prevent further infections and provide the proper treatment, rapid detection of infection is critical. However, one of the many problems faced by clinicians and the concerned public is proper methods of virus detection. Usually, a few diagnostic methods are widely accepted and used for the clinical diagnosis of patients (Dwyer et al., 2006). Although the RT-PCR method is a very accurate method, it requires expensive reagents, good facilities, and skillful operators (Amano and Cheng, 2005). Rapid antigen detection methods such as immunofluores- cence or enzyme immunoassay are simple and based on the specific binding between antigen and antibody (de Boer et al., 1990; Remarque et al., 1998). The labeling compounds typically used in these assays are the following: enzymes such as horseradish peroxidase and alkaline phosphatase, fluorophores, chemiluminescent molecules (Acridinum esters), radioisotopes like 125 I, 3 H and 57 Co, and nano- or microsized magnetic beads. Among these, enzymes have been used extensively due to their good sensitivity. The drawbacks of these enzymes are as follows: 1) several incubation and washing steps are needed. As a result the method is labour intensive. 2) The enzyme and the substrates are subject of several deactivation reactions and require well defined storage conditions. Dot-blot assay employing fluorescence compounds such as FITC is another alternative method. The method is simple and fast. The major drawback is quenching and bleaching of the fluorescence tag. As a result the method is not very robust. Accordingly, other methods not requiring enzymes and chemicals have been suggested as alternatives. Paramagnetic Journal of Immunological Methods 365 (2011) 95–100 ⁎ Corresponding author. Tel.: + 82 42 860 6663; fax: +82 42 860 5611. E-mail address: hb8868@etri.re.kr (H.-B. Hong). 0022-1759/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2010.12.005 Contents lists available at ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim