title: Nanofluidic Fluorescence Microscopy (NFM) for real-time monitoring of protein binding kinetics and affinity studies.
abstract: Kinetic monitoring of protein interactions offers insights to their corresponding functions in cellular processes. Surface plasmon resonance (SPR) is the current standard tool used for label-free kinetic assays; however, costly and sophisticated setups are required, decreasing its accessibility to research laboratories. We present a cost-effective nanofluidic-based immunosensor for low-noise real-time kinetic measurement of fluorescent-labeled protein binding. With the combination of fluorescence microscopy and reversed buffer flow operation, association and dissociation kinetics can be accessed in one single experiment without extra buffer loading step, which results in a simplified operation and reduced time of analysis compared to typical microfluidic immunoassays. Kinetic constants of two representative protein-ligand binding pairs (streptavidin/biotin; IgG/anti-IgG) were quantified. The good agreement of extracted rate constants with literature values and analogous SPR measurements indicates that this approach is applicable to study protein interactions of medium- and high-affinities with a limit of detection down to 1 pM, regardless of the analyte size.
authors: LAAS-CNRS, 7 Avenue du Colonel Roche, F-31077 Toulouse, France; Université de Toulouse, F-31077 Toulouse, France.
authors: Pattamon
authors: P
authors: Teerapanich
authors: IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM, U1194, Université Montpellier, ICM, Institut Régional du Cancer, Montpellier, F-34090 France.
authors: Martine
authors: M
authors: Pugnière
authors: IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM, U1194, Université Montpellier, ICM, Institut Régional du Cancer, Montpellier, F-34090 France.
authors: Corinne
authors: C
authors: Henriquet
authors: Institute of Physics, Academia Sinica, Taipei 11529, Taiwan.
authors: Yii-Lih
authors: YL
authors: Lin
authors: Institute of Physics, Academia Sinica, Taipei 11529, Taiwan.
authors: Chia-Fu
authors: CF
authors: Chou
authors: LAAS-CNRS, 7 Avenue du Colonel Roche, F-31077 Toulouse, France; Université de Toulouse, F-31077 Toulouse, France. Electronic address: tleichle@laas.fr.
authors: Thierry
authors: T
authors: Leïchlé
mesh_terms: D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry; D004867:Equipment Design; D007118:Immunoassay / Q000295:instrumentation; D007074:Immunoglobulin G / Q000032:analysis*; D007700:Kinetics; D051379:Mice; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D011485:Protein Binding; D019809:Streptavidin / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000295:instrumentation*
chemical_list: D055910:Antibodies, Immobilized; D007074:Immunoglobulin G; D001710:Biotin; D019809:Streptavidin
keywords: Biosensors; Fluorescence microscopy; Interaction kinetics; Nanofluidics