Detection of pathogenic Staphylococcus aureus bacteria by gold based immunosensors |
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Authors: | Souhir Boujday Romain Briandet Michèle Salmain Jean-Marie Herry Pierre-Guy Marnet Michel Gautier Claire-Marie Pradier |
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Affiliation: | 1. UPMC Univ Paris 6, UMR CNRS 7609, Laboratoire de Réactivité de Surface, F75005, Paris, France 2. CNRS, UMR 7609, Laboratoire de Réactivité de Surface, F75005, Paris, France 3. UMR 763 INRA-AgroParisTech, Bioadhésion et Hygiène des Matériaux, 25 avenue de la République, F 91300, Massy, France 4. Laboratoire de Chimie et Biochimie des Complexes Moléculaires, UMR CNRS 7576, Ecole Nationale Supérieure de Chimie de Paris, F75005, Paris, France 5. Laboratoire de microbiologie, UMR 1263 INRA-Agrocampus, 65 rue de Saint-Brieuc CS 84215, F35042, Rennes cedex, France
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Abstract: | We describe the elaboration of ultra-sensitive immunosensors, to detect the bacterial pathogen Staphylococcus aureus. We utilized commercially available polyclonal anti-S. aureus antibody as receptor. Immunosensors were elaborated by building a self-assembled monolayer (SAM) of thiolamine onto planar gold-coated sensor chips. Then, Protein A was covalently linked to the thiolated SAM using glutaraldehyde as cross-linking agent. After a blocking step by Bovine Serum Albumin (BSA), the antibody was immobilized by affinity to Protein A. This step-by-step construction was monitored by Polarization Modulation Reflection Absorption Infrared Spectroscopy (PM-RAIRS) and Quartz Crystal Microbalance with Dissipation (QCM-D). In a first stage, the parameters of immunosensor elaboration were optimized using a model rabbit IgG. The accessibility of receptors and the homogeneity of their distribution were checked by PM-RAIRS, QCM-D, and by immuno-gold scanning electron microscopy. Then, the specific rabbit anti-S. aureus antibody was immobilized and the resulting sensing layer was applied to the detection of the pathogen target. Independent detection of bacteria immobilized on the sensors by fluorescent imaging allowed validation of the specificity of recognition toward the pathogen as well as a quantitative response of the sensor. Using PM-RAIRS as transducing technique allowed us to enhance sensitivity and reach a very competitive detection level (105 CFU mL?1). |
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