FRISBI
The team of « Dynamics of Macromolecular Complexes» of the LBPA located at the ENS Cachan has been developing and using label free techniques to study macromolecular interactions involving nucleoproteins, antigen-antibody, peptide-peptide and peptide-proteins for many years. The main approach has involved Surface Plasmon Resonance (SPR) and Surface Plasmon Resonance Imagery (SPRi). The group now offers a platform that provides customised chips to study almost any type of macromolecular interaction.
SPR consists of immobilizing ligands to a surface and then observing changes in the refractive index at the surface as molecules bind. In the SPRi technique initially introduced by Genoptics (now Horiba) the biosensor surface consist of prisms made of a high refractive index material with one surface coated with a thin layer of gold. An evanescent field called a plasmon wave is created at the interface of this gold-coated surface and the dielectric from a light beam arriving through the prism at an angle of total internal reflection. At this angle there is a resonance effect that is measured by imaging the entire reflected light from a monochromatic polarized electroluminescent diode using a CCD camera linked via a dedicated optical system. This allows analysis of an entire surface upon which discrete spots of ligand have been immobilized.
Real time analysis provides information about the on (ka) and off (kd) kinetic rates for a macromolecular interaction between a molecule in solution and an immobilised ligand (and thus the Kd) and stoichiometry. A major difference between the SPR (as exemplified by BIAcore) and SPRi apart from the obvious advantage of multiplexing in the latter is that SPRi allows the development of specific monolayer surfaces. LBPA has developed a series of specific surface chemistries (General Liquid Interface Specific Surfaces –GLISS) that allow macromolecules to be favourably immobilised and orientated in a Self Assembled Monolayer that is extremely refractive to non-speciifc binding. Up to 400 independently immobilised different molecules may be interogated simultaneously on the same surface. For immobilisation each spot uses less than 2 µl of material at concentrations ranging from 0.01 to 0.1 mg/ml, and a range of amide/carboxyl/thiol coupling chemistries are available. Volumes of between 10 and 500 µl may be flowed across the surfaces at flow rates of between 10 and 500 µl/min.
The platform consists of :