DOI: 10.1002/elan.201400328 Electrochemical Aptasensor Based on Polycarboxylic Macrocycle Modified with Neutral Red for Aflatoxin B1 Detection Gennady Evtugyn, [a] Anna Porfireva, [a] Veronika Stepanova, [a] Rusal Sitdikov, [b] Ivan Stoikov, [b] Dimitrios Nikolelis, [c] and Tibor Hianik* [d] 1 Introduction Aptamers are short sequences (15–90 oligonucleotides) of single-stranded nucleic acids (DNA or RNA) that can bind with high affinity to a wide range of targets ranging from cells, viruses, proteins to low-molecular compounds [1, 2]. The aptamers are intensively investigated as biore- cognition elements for highly selective and sensitive de- tection of proteins [3–5], toxins [6–9], disease biomarkers [10] and some other compounds important for clinical di- agnostics and environmental monitoring. In comparison with antibodies, aptamers show higher stability during the storage and use as well as broad opportunities for modifi- cation and immobilization on various supports. The aptasensors, i.e. biosensors utilizing aptamers in their assembly, are mainly based on electrochemical transducers that offer many advantages like fast and relia- ble response, compatibility with conventional measure- ment equipment, prospects of miniaturization, field appli- cations and low measurement cost [11]. High specificity of the aptasensor is achieved by the ability of aptamer to fold into defined three-dimensional conformation which facilitates specific interaction with a target. The affinity constants of an analyte binding are in many cases compa- rable with those of antigen-antibody interaction. This pro- vides the quantification of analytes on sub-ppb level. Meanwhile the detection of specific aptamer interactions requires special efforts to establish a reliable and sensitive signal of appropriate transducer. Regarding electrochemi- cal aptasensors, this can be achieved by introduction of mediator systems able to decrease the working potential and amplify the response. Methylene blue [12–14], tolui- dine blue [15], methylene green [16], and ferrocene [17,18] have been successfully used for this purpose. The mediators are often combined with auxiliary materials to increase their surface concentration, compatibility with an aptamer structure and stability of the surface layer. Electropolymerized films [19–21], golden nanoparticles [22–24] and carbonaceous materials like carbon nano- tubes [25–27] and graphene [28] are mostly applied as supports in aptasensor assembly. Most of them exert their own electrochemical activity. This improves electron transduction but does not contribute to high sensitivity. Indeed, the analyte quantification is mainly based on rel- ative shift of the current recorded with modified elec- trode. In such a measurement mode, the use of electro- chemically active support increases the signal but com- pensates for the influence of binding of an analyte mole- cules. This calls for the development of new supports that combine the ability to electrochemical wiring with high sensitivity to the involvement of non-conductive species in the surface layer. Recently we have proposed a novel polycarboxylated macrocycle on thiacalix[4]arene platform as a universal support for simultaneous immobilization of aptamer and [a] G. Evtugyn, A. Porfireva, V. Stepanova Analytical Chemistry Department, Kazan Federal University 18 Kremlevskaya Street, Kazan, 420008, Russian Federation [b] R. Sitdikov, I. Stoikov Organic Chemistry Department, Kazan Federal University 18 Kremlevskaya Street, Kazan, 420008, Russian Federation [c] D. Nikolelis Laboratory of Environmental Chemistry, Department of Chemistry, University of Athens Panepistimiopolis-Kouponia, GR-15771 Athens, Greece [d] T. Hianik Department of Nuclear Physics and Biophysics, Comenius University Mlynska dolina F1, 842 48 Bratislava, Slovakia *e-mail: Tibor.Hianik@fmph.uniba.sk Abstract : Novel electrochemical aptasensors based on glassy carbon electrodes modified with electropolymer- ized Neutral red and polycarboxylated macrocyclic li- gands onto which the DNA aptamers were covalently at- tached have been developed for detection of Aflatoxin B1 (AFB1). The interaction with an analyte resulted in the decrease of the cathodic peak current of the probe measured by CV and in the increase of the electron trans- fer resistance determined by EIS. The limit of detection was found to be 0.1 nM for CV and 0.05 nM for EIS methods, respectively. The aptasensor makes it possible to detect AFB1 in peanuts, cashew nuts, white wine and soy sauce with a recovery of 85–100 %. Keywords: Aptasensor · Aflatoxin B1 · Thiacalix[4]arene · DNA Aptamer · Voltammetry · Electrochemical impedance spectroscopy www.electroanalysis.wiley-vch.de  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1 – 11 &1& These are not the final page numbers! ÞÞ Full Paper