Biosensors and Bioelectronics 27 (2011) 46–52 Contents lists available at ScienceDirect Biosensors and Bioelectronics jou rn al h om epa ge: www.elsevier.com/locate/bios Magneto immunosensor for gliadin detection in gluten-free foodstuff: Towards food safety for celiac patients T. Laube a , S.V. Kergaravat b , S.N. Fabiano b , S.R. Hernández b , S. Alegret a , M.I. Pividori a,∗ a Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia, Spain b Laboratorio de Sensores y Biosensores, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina a r t i c l e i n f o Article history: Received 7 February 2011 Received in revised form 29 May 2011 Accepted 10 June 2011 Available online 20 July 2011 Keywords: Gliadin Celiac disease Food safety Magneto-ELISA Electrochemical magneto immunosensor Direct competitive immunoassay Magnetic beads a b s t r a c t Gliadin is a constituent of the cereal protein gluten, responsible for the intolerance generated in celiac disease. Its detection is of high interest for food safety of celiac patients, since the only treatment known until now is a lifelong avoidance of this protein in the diet. Therefore, it is essential to have an easy and reliable method of analysis to control the contents in gluten-free foods. An electrochemical mag- neto immunosensor for the quantification of gliadin or small gliadin fragments in natural or pretreated food samples is described for the first time and compared to a novel magneto-ELISA system based on optical detection. The immunological reaction was performed on magnetic beads as solid support by the oriented covalent immobilization, of the protein gliadin on tosyl-activated beads. Direct, as well as indirect competitive immunoassays were optimized, achieving the best analytical performance with the direct competitive format. Excellent detection limits (in the order of g L -1 ) were achieved, according to the legislation for gluten-free products. The matrix effect, as well as the performance of the assays was successfully evaluated using spiked gluten-free foodstuffs (skimmed milk and beer), obtaining excellent recovery values in the results. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Celiac disease, also known as gluten-sensitive enteropathy, is an autoimmune disorder that affects 1:100 people in Northern Europe and North America (Lee and Green, 2006). It results from damage to the upper small intestinal mucosa due to an inap- propriate immune response towards a cereal protein, the gluten, affecting the normal absorption of many nutrients (Woodward, 2007). Gluten is the generic term used for the protein fraction of cereal grains that causes celiac disease, being present in wheat, barley and rye (Woodward, 2007). Gluten proteins have been tra- ditionally divided into roughly equal fractions according to their solubility in alcohol–water solutions (e.g. 60% ethanol): the solu- ble gliadins and the insoluble glutenins. Gliadins are monomers – with molecular weights between 30 and 70 kDa – respon- sible for triggering the immune system for the production of autoantibodies (Wieser, 2007). The treatment of celiac disease is a gluten-free diet. Therefore, gluten has been included in food labeling in order to prevent harmful effects of gluten-containing food or food components in celiac patients (Stern, 2005). The US Food and Drug Administration (FDA) defined in 2008 that foods labeled with the term gluten-free, may not exceed a gluten con- ∗ Corresponding author. Tel.: +34 93 581 4937; fax: +34 93 581 2379. E-mail address: Isabel.Pividori@uab.cat (M.I. Pividori). tent of 20 mg L -1 (Thompson and Méndez, 2008) and the same regulation has been taken in 2009 by the European Community (Commission Regulation no 41/2009, 2009). It is extremely impor- tant to have reliable methods for the detection of gliadin, not only for the celiac patients, but also for the industries generat- ing gluten-free foodstuffs in order to rapidly test incoming raw materials and check for gluten contamination throughout the food production process (Dahinden et al., 2001; Nassef et al., 2008). In consequence, a range of methods for the detection of gliadin has been reported. Classic instrumental analytical techniques – such as MALDI/TOF-MS and ES-MS combined with HPLC – (Camafeita et al., 1997 and Mamone et al., 2000) were reported, both of them requiring sophisticated equipments and trained personnel. Other strategies are based on the amplification of DNA from wheat, bar- ley and rye by polymerase chain reaction (PCR or QC-PCR) since its presence is related with the presence of the corresponding harmful antigenic proteins (Dahinden et al., 2001; Olexová et al., 2006). More recently, an optical biosensor for gliadin detection has been reported, which uses a recombinant glutamine-binding protein from Escherichia coli which is able to recognize the gliadin in micromolecular concentrations (De Stefano et al., 2006). How- ever, the most commonly used methods for gliadin measurement are based on immunological procedures, including immunoblot- ting and Enzyme Linked Immunosorbent Assay (ELISA), using monoclonal or polyclonal antibodies against a variety of gliadin components (Camafeita et al., 1997). Different ELISAs in sandwich 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.06.006