Nanofluidic Fluorescence Microscopy (NFM) for real-time monitoring of protein binding kinetics and affinity studies

Pattamon Teerapanich (1) , Martine Pugniere (2) , Corinne Henriquet (2) , Yii-Lih Lin (3) , Chia-Fu Chou (3) , Thierry Leichle (1)

1 LAAS-MEMS - Équipe Microsystèmes électromécaniques
2 IRCM - U1194 Inserm - UM - Institut de Recherche en Cancérologie de Montpellier
3 Academia Sinica

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.

Mots clés: Nanofluidics; Biosensors; Fluorescence microscopy; Interaction kinetics

Domaines: Biophysique; Micro et nanotechnologies/Microélectronique

Pattamon Teerapanich, Martine Pugniere, Corinne Henriquet, Yii-Lih Lin, Chia-Fu Chou, et al.. Nanofluidic Fluorescence Microscopy (NFM) for real-time monitoring of protein binding kinetics and affinity studies. Biosensors and Bioelectronics, 2017, 88, pp.25-33. ⟨10.1016/j.bios.2016.06.033⟩. ⟨hal-01964833⟩