Nanostructured Silver-Based Surfaces: New Emergent Methodologies for an Easy Detection of Analytes Maria Staiano, † Evgenia G. Matveeva, ‡ Mauro Rossi, § Roberta Crescenzo, † Zygmunt Gryczynski, ‡ Ignacy Gryczynski, ‡ Luisa Iozzino, †,| Irina Akopova, ‡ and Sabato D’Auria* ,† Laboratory for Molecular Sensing, Istituto di Biochimica delle Proteine-Consiglio Nazionale delle Richerche, Naples, Italy, Center for Commercialization of Fluorescence Technologies, Department of Molecular Biology and Immunology, University of North Texas, Health Science Center, Fort Worth, Texas, Institute of Food Sciences, Consiglio Nazionale delle Richerche, Avellino, Italy, and School of Veterinary Medicine, University of Naples Federico II, Naples, Italy ABSTRACT In this work, we describe how to realize a new sensing platform for an easy and fast detection of analytes. In particular, we utilized enhanced fluorescence emission on silver island films (SIFs) coupled to the total internal reflection fluorescence mode (TIRF) to develop a new assay format for the detection of target analytes. Here, as an example, we report on the detection of the toxic peptides present in gliadin (Gli). Our assay was performed as follows: (1) gliadin was first captured on surfaces coated with anti-Gli antibodies; (2) the surfaces were then incubated with fluorophore-labeled anti-Gli antibodies; (3) the signal from the fluorophore- labeled anti-Gli antibody bound to the antigen was detected by TIRF. The system was examined on glass surfaces and on SIFs. We observed a relevant enhancement of the signal from SIFs compared to the signal from the glass substrate not modified with a SIF. In addition, the estimated detection limit (EDL) of our methodology was 60 ng/mL (or lower). This limit is therefore lower than the clinical cut-off for Gli presence in food for celiac patients. The advantage of our method is a reduced number of testing steps, which allows for easy detection of residual toxic peptides in food labeled as gluten free. The proposed technology can be easily expanded to the determination of different target analytes. KEYWORDS: celiac disease • fluorescence assay • gliadin immunoassay • silver island films • total internal reflection fluorescence INTRODUCTION C eliac disease (CD; other abbreviations: area under the curve, AUC; estimated detection limit, EDL; front face, FF; gliadin, Gli; radiative decay engineering, RDE; reversed-phase HPLC, RP-HPLC; surface-enhanced Raman scattering, SERS; silver island film, SIF; total internal reflection fluorescence, TIRF; tissue transglutaminase, tTG) is an immune mediated disease of the small intestine affecting genetically susceptible people following feeding with wheat gluten and related proteins from barley and rye (1, 2). In particular, gluten is composed of two classes of proteins: gliadins, soluble in aqueous alcohols, and insoluble glutenins. Gliadin proteins are primarily responsible of CD. Reversed-phase HPLC (RP-HPLC) can separate gliadin into more than 30 components according to their polarity in ω-, R-, and γ-gliadins (3). The high content of proline residues makes gliadins very resistant to gastric, pancreatic, and intestinal proteases. This stability can play a role in the immune reactivity of gluten proteins (4). Another crucial biochemical issue involved in the pathogenesis of CD is the deamidation of specific glutamine residues of gliadin, a reaction catalysed by tissue transglutaminase (tTG) (5) that increases the ability of gliadin peptides to elicit a T-cell response (6). Taken together, these features underscore the main difficulties in developing immunoassays that are able to determine the exact content of gluten in food, especially at very low doses, in an easy way. The essential need for such methods is highlighted by the consideration that a strict gluten-free lifelong diet is mandatory for celiac patients for the recovery of the mucosal lesion. Nevertheless, full adher- ence to diet is often hampered by the very low amount of gluten required to induce the disease. In addition, uninten- tional gluten uptake can often occur through ingesting food considered gluten-free. Until now, none of the produced methods are considered to be fully adequate. Different enzyme-linked immuno-sorbent assays (ELISAs) have been developed so far to determine the gliadin content in food. Some of them are based on the detection of the heat- stable ω-gliadin (7) whose content in the gliadin mixture is quite variable, depending on the different cultivars. More * Correspondence to: Dr. Sabato D’Auria, Laboratory for Molecular Sensing, IBP-CNR, Via Pietro Castellino, 111, 80131 Napoli, Italy, Tel: +39-0816132250, Fax: +39-0816132277, E-mail: s.dauria@ibp.cnr.it. Received for review September 11, 2009 and accepted December 02, 2009 † Laboratory for Molecular Sensing, Istituto di Biochimica delle Proteine-Consiglio Nazionale delle Richerche. ‡ University of North Texas. § Institute of Food Sciences, Consiglio Nazionale delle Richerche. | University of Naples Federico II. DOI: 10.1021/am900617p © 2009 American Chemical Society ARTICLE www.acsami.org VOL. 1 • NO. 12 • 2909–2916 • 2009 2909 Published on Web 12/09/2009