Impedimetric DNA Aptasensor for Sensitive Detection of Ochratoxin A in Food Gabriela Castillo, a Ilaria Lamberti, b Lucia Mosiello, c Tibor Hianik* a a Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynska dolina F1, 84248 Bratislava, Slovakia b University of Roma, Tre Department of Biology, Viale G. Marconi 446, 00149 Roma, Italy c ENEA, Italian National Agency for New Technologies, Energy and the Environment, Via Anguillarese 301, 00060 Roma, Italy *e-mail: tibor.hianik@fmph.uniba.sk Received: August 31, 2011; & Accepted: October 13, 2011 Abstract We report a high sensitive biosensor based on DNA aptamers for detection of ochratoxin A (OTA). The thiolated DNA aptamers specific to OTA of different configurations have been immobilized by chemisorption to the surface of a gold electrode. Electrochemical impedance spectroscopy (EIS) in the presence of a redox probe [Fe(CN) 6 ] 3/4 has been used for the determination of the charge transfer resistance, R ct , followed by addition of OTA. The R ct in- creased with increasing OTA concentration in the range of 0.1–100 nM. The limit of detection (0.12–0.40 nM) de- pended on the configuration of the aptamers. The sensor was regenerable and validated in food samples with satis- factory recovery. Keywords: DNA aptamer, Ochratoxin A, Electrochemical impedance spectroscopy, Biosensors DOI: 10.1002/elan.201100485 1 Introduction Electrochemical biosensors based on DNA aptamers (ap- tasensors) are of growing interest due to their high sensi- tivity and selectivity [1–5]. This is particularly due to unique properties of DNA or RNA aptamers – the single stranded nucleic acids with high affinity to proteins or to other low and macromolecular compounds, which is com- parable with that of antibodies. In contrast to antibodies, aptamers are synthesized in vitro by the SELEX proce- dure [6,7]. Aptamers also present high versatility to being chemically functionalized by biotin, thiol or amino groups, allowing them to be immobilized on various solid supports. In contrast with antibodies aptamers are more stable and the aptasensors can also be regenerated. These special characteristics open new routes in biosensor as- sembling for further practical applications, such as diag- nosis purposes in medicine or detection of toxicants in food or environment. Recently a DNA aptamer sensitive to mycotoxin – ochratoxin A (OTA) – has been devel- oped [8]. OTA is a toxical fungal metabolite produced by Aspergillus ochraceus and Penicillium verrucosum that has been identified as a contaminant in cereals, coffee, cocoa, dried fruits, pork and occasionally in the field of grapes, during storage. It may also be present in blood and kidneys of animals that have been fed contaminated foodstuff. Animal studies indicated that OTA is carcino- genic [9]. Already in 1991 the Joint FAO/WHO Expert Committee on Food Additives (JECFA) evaluated a pro- visional tolerable weekly intake (PTWI) of 112 ng/kg body weight (b.w.) for OTA. This evaluation was based on porcine nephropathies data [9]. Most recently, the Eu- ropean Commission has fixed the maximum concentra- tion of OTA in foodstuffs to: 3 mg/kg (7.4 nM) for cereal products, 5 mg/kg (12.4 nM) for roasted coffee and up to 10 mg/kg (25 nM) for instant coffee. A similar contamina- tion limit was fixed for dry grapes (10 mg/kg) (EC No. 466/2001, 1881/2006), but a contamination limit of 2 mg/L (5 nM) is valid for wine (EC No. 123/2005). An even stronger limit of OTA contamination was fixed for all baby food (0.5 mg/kg, EC No. 466/2001). Cereals, especial- ly in countries with hot climate have a strong tendency to be contaminated by OTA. Many works also reported con- tamination of grape juices with approximately 7 mg/L OTA (see [9] and references herein). The development of fast and efficient methods for OTA detection is therefore of significant importance. Biosensors for OTA determina- tion developed so far were based particularly on the oxi- dation of OTA at glassy carbon electrodes (limit of detec- tion (LOD) 0.26 mM) [10], at cysteamine self-assembled monolayers (LOD = 10 pM) [11] or reduction of horse- radish peroxidase (LOD = 0.25 nM) [12]. The indirect amperometric immunoassay utilized polyclonal antibodies against OTA immobilized on screen-printed electrodes [13] or gold colloid was of similar LOD (0.2 nM) [14]. A negligible matrix effect was observed for detection of OTA in corn samples. Improved sensitivity has been ob- tained in indirect amperometric immunoassay in which polyclonal antibodies were immobilized on dextran modi- fied gold screen-printed electrodes (LOD = 0.12 nM). The TOPICAL CLUSTER 512 # 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2012, 24, No. 3, 512 – 520 Full Paper