Selection of aptamers against Ara h 1 protein for FO-SPR biosensing of peanut allergens in food matrices Dinh T. Tran 1,2 , Karel Knez 1 , Kris P. Janssen, Jeroen Pollet 3 , Dragana Spasic, Jeroen Lammertyn n BIOSYST-MeBioS, KU Leuven–University of Leuven, Willem de Croylaan 42, B-3001 Leuven, Belgium article info Article history: Received 2 October 2012 Received in revised form 21 November 2012 Accepted 4 December 2012 Available online 20 December 2012 Keywords: Aptamer Peanut allergen Ara h 1 Capillary electrophoresis (CE) Systematic evolution of ligands by exponential enrichment (SELEX) Fiber optic surface plasmon resonance (FO-SPR) abstract The rising prevalence to food allergies in the past two decades, together with the fact that the only existing therapy is avoidance of allergen-containing food next to the implementation of anti-allergic drugs, urges the need for improved performance of current assays to detect potential allergens in food products. Therein, the focus has been on aptamer-based biosensors in recent years. In this paper we report for the first time the selection of aptamers against one of the most important peanut allergens, Ara h 1. Several Ara h1 DNA aptamers were selected after eight selection rounds using capillary electrophoresis (CE)-SELEX. The selected aptamers specifically recognized Ara h 1 and did not significantly bind with other proteins, including another peanut allergen Ara h 2. The dissociation constant of a best performing aptamer was in the nanomolar range as determined independently by three different approaches, which are surface plasmon resonance, fluorescence anisotropy, and capillary electrophoresis (353 782 nM, 419 763 nM, and 450 760 nM, respectively). Furthermore, the selected aptamer was used for bioassay development on a home-built fiber optic surface plasmon resonance (FO-SPR) biosensor platform for detecting Ara h 1 protein in both buffer and food matrix samples demonstrating its real potential for the development of novel, more accurate aptamer-based biosensors. In conclusion, the reported aptamer holds a great potential for the detection of Ara h 1 in both the medical field and the food sector due to its high affinity and specificity for the target protein. & 2012 Elsevier B.V. All rights reserved. 1. Introduction Peanut allergy is one of the most serious, life-threatening food sensitivities since it triggers the highest frequency of severe and fatal reactions. It represents a lifelong disorder with high risk of accidental exposures (Kleber-Janke et al., 1999). Ara h 1 is considered one of the major peanut allergens. It is a glycoprotein with a molecular weight of 63,000 Da and a pI of 4.5 (Burks et al., 1991). Ara h 1 exists in food as a homotrimeric complex. It has been shown that this particular tertiary structure contributes to the high allergenicity of the protein even after enzymatic and heat treatments. Thus, domains critical for oligomerization coincide with various protease-resistant fragments that protect molecule from protease digestions and denaturation. Because these domains also contain IgE recognition sites within tertiary struc- ture, numerous of them can survive as large Ara h 1 fragments during gastrointestinal degradation of protein, which is a pre- requisite for triggering high titer of IgE and allergenic responses (Maleki et al., 2000). Because the frequency of sensitization by Ara h 1 can be up to 100% of peanut allergic patients (Kleber-Janke et al., 1999), it is used as a marker for monitoring peanut contamination of food products. For these reasons, the development of improved detection methods for Ara h 1 remains priority in ensuring the compliance of food labeling and consumer protection. To date, several immuno-assay techniques have been described in literature for detection of Ara h 1 including dip stick assay (Mills et al., 1997), ELISA (Pomes et al., 2004, lateral flow assay (Wen et al., 2005), and immunosensors (Huang et al., 2008; Pollet et al. 2011). While these methods are superior in either sensitivity, ease of use, or required assay time, they all rely on antibodies which are sometimes relatively difficult to generate (for instance antibodies against molecules that animals cannot tolerate, such as toxins) (Jayasena, 1999). Moreover, antibodies Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics 0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.12.022 n Corresponding author. Tel.: þ32 16 321459; fax: þ32 16 322955. E-mail addresses: ttdinh@hua.edu.vn (D.T. Tran), Karel.Knez@biw.kuleuven.be (K. Knez), kris.janssen@biw.kuleuven.be (K.P. Janssen), jeroen.pollet.biosensors@gmail.com (J. Pollet), Dragana.Spasic@biw.kuleuven.be (D. Spasic), jeroen.lammertyn@biw.kuleuven.be (J. Lammertyn). 1 Both authors equally contributed to this manuscript. 2 Presnt address: Faculty of Food Science and Technology, Hanoi University of Agriculture, 10000 Hanoi, Vietnam. 3 Present address: Sabin Vaccine Institute and Texas Children’s Hospital Center for Vaccine Development, Department of Pediatrics, Section of Tropical Medicine, Baylor College of Medicine, Houston, TX 77030, USA. Biosensors and Bioelectronics 43 (2013) 245–251