Immunodetection of 17b-estradiol in serum at ppt level by microcantilever resonators Carlo Ricciardi a,n , Ivan Ferrante a , Riccardo Castagna a , Francesca Frascella a , Simone Luigi Marasso a,b , Karin Santoro a , Marilena Gili c , Danilo Pitardi c , Marzia Pezzolato c , Elena Bozzetta c a Politecnico di Torino—LATEMAR Unit, Department of Applied Science and Technology, Corso Duca degli Abruzzi 24, I-10129 Torino, Italy b Trustech s.r.l. @ Techfab micro & nano technologies center, Localit  a Baraggino, 10034 Chivasso, Italy c Istituto Zooprofilattico Sperimentale di Piemonte, Liguria e Valle d 0 Aosta, Via Bologna 148, 10154 Torino, Italy article info Available online 29 August 2012 Keywords: 17b-estradiol Microcantilever Immunodetection Nanobiosensors Public health abstract To date control strategies in detecting anabolic agents for promoting growth of food producing animals are mainly related to screening techniques based on immunochemical and physiochemical methods, whose major limit is represented by relative low analytical sensitivity. As a consequence, consumers are currently exposed to molecules with potential carcinogenic effects such as 17b-estradiol, the most powerful substance with estrogenic effect. Therefore, high analytical sensitivity screening and confirmatory methods are required, coupling easiness of use and efficiency. We here report on the immunodetection of 17b-estradiol in serum by antibody-immobilized microcantilever resonators, an innovative biosensing platform able to quantify an adsorbed target mass (such as cells, nucleic acids, biomolecules, etc.) thanks to a shift in resonance frequency. Our tool based on microcantilever resonator arrays has shown to be capable of discriminating treated and untreated animals, showing the ability of detecting traces of 17b-estradiol in serum at concentrations lower than the present accepted physiological serum concentration threshold value (40 ppt) and commercial ELISA tests (25 ppt). The method exhibits a limit of detection of 20 ppt and a limited cross-reactivity with high concentrations (10 ppb) of similar molecules (testosterone). & 2012 Elsevier B.V. All rights reserved. 1. Introduction Anabolic agents for promoting growth of food producing animals have been widely used over the last past 50 years due to their action on lipid and protein metabolism, increasing of growth rate, muscle growth stimulation and reduction of fat tissue (Groot et al., 1998). One of the most efficient of this anabolics is 17b-estradiol, that is involved in a number of physiological processes, including development of the female reproductive structures and secondary sexual characteristics, mineral, fat, sugar and protein metabolism, intestinal motility, blood coagulation, cholesterol metabolism, sodium and water conservation by the kidneys (Noppe et al., 2008). However, besides these functions, it is a potential carcinogen and its use in food producing animals has been banned since 2008 by the European Parliament and the Council of the European Union (Directive 2008/97/EC). Notwithstanding the intensive control of illegal growth promoters within the EU, the number of reported positives in cattle in the last few years was very limited. This may be due to the combination of very low doses of different drugs (cocktails) (Courthney et al., 2002), causing difficulties in their detection in matrices of biological origin. To date control strate- gies are mainly related to screening techniques based on immu- nochemical and physiochemical methods (Gardini et al., 2006), whose major limit is represented by relative low analytical sensitivity. As a consequence, the present accepted physiological serum concentration for this natural hormone is fixed at 40 ppt (part-per-trillion) in the Italian Residue Control Plan (Ministerial Decree 14 November 1996) to precaution include physiological variability in different bovine category (calves, bulls, heifers), while when screening systems with higher performance would be available, the threshold of positivity could be fixed at lower levels and related to the specific animal category. This approach would allow fulfilling the requirement of ‘‘zero tolerance’’ in edible matrices, thus avoiding the ongoing exposure of consumers to residues of this unauthorized molecule. Therefore, high analytical sensitivity screening and confirmatory methods are required (De Brabander et al., 2007; Groot et al., 2000), coupling easiness of use and efficiency. Microcantilever-based mass resonators, with selective coat- ings for target immobilization, represent one of the most promis- ing classes of label free biosensor platforms (Waggoner and 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.2012.08.043 n Corresponding author. Tel.: þ39 11 0907398; fax: þ39 11 0907399. E-mail address: carlo.ricciardi@polito.it (C. Ricciardi). Biosensors and Bioelectronics 40 (2013) 407–411