A fluorescent aptasensor for sensitive analysis oxytetracycline based on silver nanoclusters Morteza Hosseini, a * Fatemeh Mehrabi, a Mohammad Reza Ganjali b,c and Parviz Norouzi b,c ABSTRACT: A fluorescent aptasensor for detection of oxytetracycline (OTC) was presented based on fluorescence quenching of DNA aptamer-templated silver nanoclusters (AgNCs). The specific DNA scaffolds with two different nucleotides fragments were used: one was enriched with a cytosine sequence fragment (C12) that could produce DNA–AgNCs via a chemical reduction method, and another was the OTC aptamer fragment that could selectively bind to the OTC antibiotic. Thus, the as-prepared AgNCs could exhibit quenched fluorescence after binding to the target OTC. The fluorescence ratio of the DNA–AgNCs was quenched in a linearly proportional manner to the concentration of the target in the range of 0.5 nM to 100 nM with a detection limit of 0.1 nM. This proposed nanobiosensor was demonstrated to be sensitive, selective, and simple, introducing a viable alter- native for rapid determination of toxin OTC in honey and water samples. Copyright © 2016 John Wiley & Sons, Ltd. Keywords: oxytetracycline; silver nanoclusters; fluorescence; quenching; aptamer; nanobiosensor Introduction Antibiotics are a class of compounds with antibacterial activity, that is they have the ability to kill or inhibit the growth of bacteria (1). During the past few decades, oxytetracycline (OTC), one of the broad-spectrum antibacterial tetracyclines (TCs), has been widely used in agriculture and animal husbandry as a bacterial infection inhibitor or growth promoter. It is characterized by the exceptional chemotherapeutic efficacy against a wide range of Gram-positive and Gram-negative bacteria based on the inhibition of bacterial protein synthesis (2). However, due to its frequent abuse, the OTC residue in the environment or its accumulation in food has caused a series of negative effects such as drug resistance in bac- teria and allergic reactions in the human body (3–5). Therefore, a simple and effective approach to detect OTC in contaminated food products is urgently needed. Traditionally, chromatography methods, including high-performance liquid chromatography (HPLC) and liquid chromatography- tandem mass spectrometry (LC–ESI-MS/MS), have been used for the detection of TCs in food products (6,7). Although these methods provide simultaneous and accurate detection of TCs, they demand expensive equipment, tedious sample extraction procedures, and expert technical skills. Moreover, immunochem- ical methods based on antibodies have been used owing to their simplicity and cost–effectiveness and detection with high sensi- tivity and specificity (8), but the use of an antibody as a probe for detection has some drawbacks in terms of stability and production. Aptamers are the main specific oligonucleotides (ssDNA or RNA) for capture and detection of a wide range of targets that are obtained by an in vitro selection process called systematic evolution of ligands by exponential enrichment (SELEX). Compared with antibodies, aptamers are easily synthesized, modified and fixed, repeatedly used, and preserved long term (9,10). Therefore, aptamers have been widely used as recognition elements to make aptamer-based biosensors, which have been applied in protein research (11) and mycotoxins (12,13). Since Niazi et al. selected a DNA aptamer that can be used to specifically detect OTC in 2008 (14), numerous studies have been reported for the development of an aptamer-based biosensor for OTC analysis (15) based on electrochemical, colorimetric, light scatter- ing or microcantilever methods (16). Although they have success- fully realized quantitative analysis of OTC, many concomitant limitations still exist. Recently, there has been an explosion of interest in fluorescent silver nanocluster (AgNC) synthesis and their application in the area of bioassays (17). AgNCs with a few atoms, exhibiting size-dependent fluorescence emission, have been developed as a new class of fluorophores. The synthesis of fluorescent AgNCs using DNA as scaffolds in aqueous solution has attracted exten- sive attention (18). The DNA scaffolded AgNCs (DNA–AgNCs) exhibit outstanding spectral and photophysical properties, and the photoluminescence (PL) emission band can be fine tuned just by changing the sequence of DNA (19). They display excel- lent photostability, subnanometer size, nontoxicity, biocompati- bility and are thus well suited as a fluorescent probe for * Correspondence to: M. Hosseini, Department of Life Science Engineering, Faculty of New Sciences & Technologies, University of Tehran, Tehran, Iran. Tel: +98 21 61112788; Fax: +98 21 61112788. E-mail: smhosseini@khayam. ut.ac.ir a Department of Life Science Engineering, Faculty of New Sciences & Technolo- gies, University of Tehran, Tehran, Iran b Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran, Iran c Biosensor Research Center, Endocrinology & Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran Abbreviations: HPLC, high-pressure liquid chromatography; TEM, transmis- sion electron microscopy Luminescence 2016 Copyright © 2016 John Wiley & Sons, Ltd. Research article Received: 16 November 2015, Revised: 10 January 2016, Accepted: 17 January 2016 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/bio.3112