Multiclonal plastic antibodies for selective aflatoxin extraction from food samples Engin Bayram a , Erkut Yılmaz a , Lokman Uzun a,⇑ , Rıdvan Say b,c , Adil Denizli a a Hacettepe University, Department of Chemistry, Ankara, Turkey b Anadolu University, Department of Chemistry, Eskis ßehir, Turkey c Bionkit Ltd. S ßti, Eskis ßehir, Turkey article info Article history: Received 27 February 2016 Received in revised form 10 November 2016 Accepted 20 November 2016 Available online xxxx Keywords: Molecular imprinting Multiclonal plastic antibodies Simultaneous imprinting Cryogels Aflatoxin extraction Food samples abstract Herein, we focused on developing a new generation of monolithic columns for extracting aflatoxin from real food samples by combining the superior features of molecularly imprinted polymers and cryogels. To accomplish this, we designed multiclonal plastic antibodies through simultaneous imprinting of aflatoxin subtypes B1, B2, G1, and G2. We applied Fourier transform infrared (FTIR) spectroscopy, scanning elec- tron microscopy (SEM), and spectrofluorimetry to characterize the materials, and conducted selectivity studies using ochratoxin A and aflatoxin M1 (a metabolite of aflatoxin B1), as well as other aflatoxins, under competitive conditions. We determined optimal aflatoxin extraction conditions in terms of con- centration, flow rate, temperature, and embedded particle amount as up to 25 ng/mL for each species, 0.43 mL/min, 7.0, 30 °C, and 200 mg, respectively. These multiclonal plastic antibodies showed imprint- ing efficiencies against ochratoxin A and aflatoxin M1 of 1.84 and 26.39, respectively, even under com- petitive conditions. Finally, we tested reusability, repeatability, reproducibility, and robustness of columns throughout inter- and intra-column variation studies. Ó 2016 Elsevier Ltd. All rights reserved. 1. Introduction Whenever mycotoxins are mentioned, aflatoxin is the first one that comes to mind because it has been intensively exam- ined. Aflatoxins are synthesized by Aspergillus flavus, Aspergillus parasiticus, and some subspecies of Aspergillus nomius (Chiavaro et al., 2001). These fungal metabolites, mainly consisting of afla- toxin B1, B2, G1, G2, M1, and M2, may acutely and/or chroni- cally poison humans and animals depending on the exposure dose and duration (Jaimez et al., 2000; Ren et al., 2007; Tanaka et al., 2015). Acute exposure in animals directly affects the liver (Bryden, 2012) and results in other symptoms such as anorexia, hemolytic anemia, diarrhea, and icterus (Bbosa et al., 2013; Tang, Guan, Ding, & Wang, 2007; Trebak et al., 2015). Moreover, the resulting liver damage triggers cells in the liver and gall channels to rapidly proliferate, leading to cramps, paral- ysis, balance disorders, and hemorrhaging; furthermore, the ner- vous system may also be affected (Trebak et al., 2015). Recently, the International Agency for Research on Cancer (IARC), a part of the World Health Organization, initiated many studies regarding the effects of aflatoxins on human health and defined aflatoxins as Group 1 Carcinogenic Substances in light of the results (Corcuera, Vettorazzi, Arbillaga, Gonzalez-Penas, & Lopez de Cerain, 2012; IARC, 2012; Frehse et al., 2015; Trebak et al., 2015). In Turkey and many other countries, authorities have imposed regulations to control aflatoxin levels (especially B1) in food and forage, as aflatoxin levels (especially M1) in milk to protect consumers, especially children, from contaminated products (TR Food Codex., 2011). Today, almost all countries have assigned maximum aflatoxin levels in food and forage, not only to protect themselves, but also to avoid economic loss from the retrocession of products during international trade (Akiyama, Goda, Tanaka, & Toyoda, 2001; Bryden, 2012). The constant demand for new, fast, and efficient methods in environmental, medicine, and biotechnology fields has guided scientists to develop more selective and sensitive alternative detection methods. Among separation systems, molecular recognition-based carriers prepared via molecular imprinting have become more prominent due to their high selectivity for target molecules (Alexander et al., 2006; de Smet et al., 2010; Uzun & Turner, 2016). Molecular imprinting, a unique polymer- ization technique, is used for obtaining highly selective binding cavities through a three-dimensional (3D) orientation of func- tional groups around the target molecule (generally called the http://dx.doi.org/10.1016/j.foodchem.2016.11.090 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved. ⇑ Corresponding author at: Hacettepe University, Department of Chemistry, Biochemistry Division, Ankara, Turkey. E-mail address: lokman@hacettepe.edu.tr (L. Uzun). Food Chemistry xxx (2016) xxx–xxx Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Please cite this article in press as: Bayram, E., et al. Multiclonal plastic antibodies for selective aflatoxin extraction from food samples. Food Chemistry (2016), http://dx.doi.org/10.1016/j.foodchem.2016.11.090