Electrophoresis 2012, 33, 3195–3204 3195 Ludmila Krejcova 1 Dana Dospivova 1 Marketa Ryvolova 1,2 Pavel Kopel 1,2 David Hynek 1,2 Sona Krizkova 1,2 Jaromir Hubalek 1,2 Vojtech Adam 1,2 Rene Kizek 1,2 1 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Czech Republic, European Union 2 Central European Institute of Technology, Brno University of Technology, Czech Republic, European Union Received April 15, 2012 Revised July 2, 2012 Accepted July 3, 2012 Research Article Paramagnetic particles coupled with an automated flow injection analysis as a tool for influenza viral protein detection Currently, the influenza virus infects millions of individuals every year. Since the influenza virus represents one of the greatest threats, it is necessary to develop a diagnostic tech- nique that can quickly, inexpensively, and accurately detect the virus to effectively treat and control seasonal and pandemic strains. This study presents an alternative to current detection methods. The flow-injection analysis-based biosensor, which can rapidly and eco- nomically analyze a wide panel of influenza virus strains by using paramagnetic particles modified with glycan, can selectively bind to specific viral A/H5N1/Vietnam/1203/2004 protein-labeled quantum dots. Optimized detection of cadmium sulfide quantum dots (CdS QDs)-protein complexes connected to paramagnetic microbeads was performed us- ing differential pulse voltammetry on the surface of a hanging mercury drop electrode (HMDE) and/or glassy carbon electrode (GCE). Detection limit (3 S/N) estimations based on cadmium(II) ions quantification were 0.1 g/mL or 10 g/mL viral protein at HMDE or GCE, respectively. Viral protein detection was directly determined using differential pulse voltammetry Brdicka reaction. The limit detection (3 S/N) of viral protein was estimated as 0.1 g/mL. Streptavidin-modified paramagnetic particles were mixed with biotinylated se- lective glycan to modify their surfaces. Under optimized conditions (250 g/mL of glycan, 30-min long interaction with viral protein, 25°C and 400 rpm), the viral protein labeled with quantum dots was selectively isolated and its cadmium(II) content was determined. Cadmium was present in detectable amounts of 10 ng per mg of protein. Using this method, submicrogram concentrations of viral proteins can be identified. Keywords: Electrochemical detection / Hemagglutinin / Influenza / Magnetic separation / Voltammetry DOI 10.1002/elps.201200304 1 Introduction Influenza represents one of the greatest threats in soci- ety today. It is not surprising that the World Health Or- ganization (WHO) initiated the Global Influenza Program, which provides member states with strategic guidance, tech- nical support, and coordination of activities essential to bet- ter prepare healthcare systems against seasonal, zoonotic, and pandemic influenza threats to populations and individ- uals (http://www.who.int/influenza/en/) [1, 2]. According to WHO, seasonal influenza is responsible for several million Correspondence: Professor Rene Kizek, Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ- 613 00 Brno, Czech Republic E-mail: kizek@sci.muni.cz Fax: +420-5-4521-2044 Abbreviations: CdS QDs, cadmium sulfide quantum dots; FIA, flow injection analysis; GCE, glassy carbon electrode; HA, hemagglutinin; HMDE, hanging mercury drop electrode; QD, quantum dots; WHO, World Health Organization cases of acute illnesses and between 250 000 and 500 000 deaths each year [3]. There are three genera of the influenza virus: Influenza virus A, Influenza virus B, and Influenza virus C. They are RNA viruses that make up three of the five genera of the Or- thomyxoviridae family. Type A virus, the most virulent human pathogens among the three influenza types, causes the most severe disease. This virus can be further subdivided into dif- ferent serotypes based on antibody responses. The influenza virus contains two major surface proteins: hemagglutinin (HA) and neuraminidase. HA mediates glycan receptor bind- ing and membrane fusion for viral entry. Neuraminidase conducts receptor-destroying enzyme activity important for virus release [4]. Subtypes of influenza viruses are classified according to these proteins [5, 6]. More than 500 cases of avian H5N1 influenza infections in humans have been reported from 15 countries, of which nearly 60% have resulted in death (Fig. 1). Recently published meta-analysis in Science shows 12 677 participants in 20 stud- ies that have been infected with the avian H5N1 have mild Colour Online: See the article online to view Figs. 1–8 in colour. C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com