Direct fluorimetric sensing of UV-excited analytes in biological and environmental samples using molecularly imprinted polymer nanoparticles and fluorescence polarization Xuan-Anh Ton a , Victor Acha b , Karsten Haupt a,n , Bernadette Tse Sum Bui a,n a Compi  egne University of Technology, UMR CNRS 6022, B.P. 20529, 60205 Compi  egne cedex, France b Institut Polytechnique LaSalle Beauvais, B.P 30313, 60026 Beauvais Cedex, France article info Article history: Received 20 December 2011 Received in revised form 1 March 2012 Accepted 21 March 2012 Available online 10 April 2012 Keywords: Molecularly imprinted polymer Fluoroquinolones Biosensing Milk Fluorescence polarization Biomimicry abstract A rapid, robust, sensitive and economic sensing method, based on a molecularly imprinted polymer (MIP) synthetic antibody mimic, and fluorescence polarization analysis, for the direct detection of UV-excited fluorescent analytes in food and environmental samples was developed. Fluoroquinolone (FQ) antibiotics were used as fluorescent model analytes. Water-compatible MIP nanoparticles were synthesized with enrofloxacin (ENRO) as the imprinting template. Fluorescence polarization measure- ments then allow the direct determination of the amount of ENRO and other structurally related piperazine-based fluoroquinolones that bind to the MIP. No separation step was required since this technique distinguishes in situ analyte molecules bound to the MIP from the free analyte in solution. This assay was successfully applied for the first time to determine FQs in real samples, i.e. tap water and milk, without any prior concentration step, by simply adding a known amount of MIP. No interference by the sample components was observed even though the excitation was in the UV region. In tap water, a low limit of detection of 0.1 nM for ENRO was achieved with 5 mg mL 1 of MIP. In milk, ENRO and danofloxacin, whose MRLs have been fixed at 0.28 mM and 0.08 mM, respectively, could be selectively measured and distinguished from other families of antibiotics. The procedure is very easy and practical as it consists of simply precipitating the milk proteins with acetonitrile and adding buffer and MIP to the supernatant before reading the polarization values with a spectrofluorimeter. & 2012 Elsevier B.V. All rights reserved. 1. Introduction Fluorescence spectroscopy is a very sensitive method for the detection of fluorescent analytes, for example certain antibiotics, mycotoxins and other contaminants like polyaromatic hydrocar- bons, in food and environmental samples. However, fluorescence methods are also sensitive to interferences present in the sample, therefore direct measurements are often impossible, in particular if the fluorophore is excited in the UV range. Thus, a time-consuming sample preparation method normally has to be applied followed by separation before the detection of the fluorophore. In this paper, we investigate the possibility to directly determine such analytes in complex samples like milk, by providing selectivity through a molecularly imprinted polymer, and by using fluorescence polar- ization in order to specifically measure only analyte bound to the polymer. As a model analyte, we have chosen the fluoroquinolone (FQ) antibiotic enrofloxacin (ENRO). Fluoroquinolones (Fig. 1) are widely used nowadays in human and veterinary medicine. Their extensive use has led to their persistence in foodstuffs of animal origin as well as in different environmental compartments. Concerns have emerged about the effect of these residues on human health because of the serious problems induced, such as the development of antibiotic-resistant bacteria or allergic reactions. Therefore, for safety reasons, maximum residue limits (MRLs) have been established by the Council Regulation (EEC) no. 2377/90 (2000) of the European Union. The MRL of ENRO in milk has been fixed to 100 mg kg 1 . No specific limits for antibiotics in environmental waters have been established but the threshold values for residues of veterinary drugs in ground waters has been set to 0.1 mgL 1 by the 1996–1997 guideline of the European Agency for the Evaluation of Medicinal products (EMEA) (Sturini et al., 2010). Due to the low concentration level of FQs to be analyzed, a sample pretreatment step involving a solid phase extraction (SPE) on anion- exchange resins or reversed-phased cartridges, or on more specific materials like molecularly imprinted polymers (MIPs) that afford cleaner extraction (Pichon and Haupt, 2006) is usually required. 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.2012.03.033 n Corresponding authors. Tel.: þ33 344234402; fax: þ33 344203910. E-mail addresses: xuan-anh.ton@utc.fr (X.-A. Ton), Victor.acha@lasalle-beauvais.fr (V. Acha), karsten.haupt@utc.fr (K. Haupt), jeanne.tse-sum-bui@utc.fr (B. Tse Sum Bui). Biosensors and Bioelectronics 36 (2012) 22–28