ORIGINAL PAPER Analytical performances of a DNA-ligand system using time-resolved fluorescence for the determination of ochratoxin A in wheat Annalisa De Girolamo & Linda Le & Gregory Penner & Roberto Schena & Angelo Visconti Received: 4 February 2012 / Revised: 16 April 2012 / Accepted: 24 April 2012 / Published online: 12 May 2012 # Springer-Verlag 2012 Abstract The analytical performances of a novel DNA- ligand system using the time-resolved fluorescence (TRF) response of ochratoxin A (OTA)–terbium–DNA aptamer interaction were tested for the quantitative determination of OTA in wheat. Wheat was extracted with acetonitrile/ water (60:40, v/v) followed by clean-up through affinity columns containing a DNA-aptamer-based oligosorbent. Then, OTA was detected by TRF spectroscopy after reaction with a terbium fluorescent solution containing the DNA- aptamer probe. The entire procedure was performed in less than 30 min, including sample preparation, and allowed analysis of several samples simultaneously with a 96-well microplate reader. The average recovery from samples spiked with 2.5-25 μg kg -1 OTA was 77 %, with a relative standard deviation lower than 6 % and a quantification limit of 0.5 μg kg -1 . Comparative analyses of 29 naturally con- taminated (up to 14 μg kg -1 ) wheat samples using the aptamer-affinity column/TRF method or the immunoaffinity column/high-performance liquid chromatography method showed good correlation (r 0 0.985) in the range tested. The trueness of the aptamer-based method was additionally assessed by analysis of two quality control wheat materials for OTA. The DNA-ligand system is innovative, simple and rapid, and can be used to screen large quantities of samples for OTA contamination at levels below the EU regulatory limit with analytical performances satisfying EU criteria for method acceptability. Keywords Ochratoxin A . DNA aptamer . Aptamer-affinity columns . Time-resolved fluorescence . Terbium . Wheat Introduction Ochratoxin A (OTA) is a mycotoxin produced by Pen- icillium and Aspergillus moulds and can be found in a wide range of foods, such as cereals, beer, wine, cocoa, coffee, dried vine fruit and spices, and in meat products as a result of contamination of animal feed [1]. OTA is nephrotoxic and carcinogenic and poses a serious threat to the health of both humans and animals [2, 3]. The International Agency for Research on Cancer has clas- sified OTA as a possible carcinogen for humans (group 2B) [4]. The Joint FAO/WHO Expert Committee on Food Additives estimated that cereals and cereal prod- ucts as food commodities contribute more than 50 % to human OTA exposure [2]. The European Commission has set maximum limits for OTA in raw cereal grains, roasted coffee and coffee products (5 μg kg -1 ), products derived from cereals (3 μg kg -1 ), and other food com- modities, including dried fruits, grape juice, wines, spices, and liquorice root [5, 6]. Analytical methods for the detection of OTA, alone or in combination with other mycotoxins, have been reviewed [7–10]. Immunoaffinity column (IAC) clean-up in combination with high-performance liquid chromatog- raphy (HPLC) with fluorescence detection (FLD) is the most widely used procedure for the determination of OTA in food and feed. IAC offers several advantages, including provision of clean extracts from complex matrices owing A. De Girolamo (*) : R. Schena : A. Visconti Institute of Sciences of Food Production (ISPA), National Research Council of Italy, Via G. Amendola 122/O, 70126 Bari, Italy e-mail: annalisa.degirolamo@ispa.cnr.it L. Le : G. Penner NeoVentures Biotechnology Inc, 516 Colborne Street, London, ON N6B 2T5, Canada Anal Bioanal Chem (2012) 403:2627–2634 DOI 10.1007/s00216-012-6076-6