Ligand-receptor interactions in complex media: a new type of biosensors for the detection of coagulation factor VIII.

Detection of receptor-ligand interaction in complex media remains a challenging issue. We report experimental results demonstrating the specific detection of the coagulation factor VIII in the presence of a large excess of other proteins using the new BIA-ATR technology based on attenuated total reflection Fourier transform infrared spectroscopy. The principle of the detection is related to the ability of factor VIII molecules to bind to lipid membranes containing at least 8% phosphatidylserine. Several therapeutic concentrates of factor VIII were analyzed and the binding of the coagulation factor was monitored as a function of time. We show that a non-specific adsorption of stabilizing agents (typically, von Willebrand factor and human serum albumin) may be avoided by controlling the geometry of the ATR element. A linear response of the sensors as a function of the factor VIII concentration is described for different lipid membrane compositions.

Biochemical interaction analysis on ATR devices: a wet chemistry approach for surface functionalization.

A new generic device suitable for the investigation of ligand-receptor interactions is presented. In particular, the research focused on optical waveguides constituted by an attenuated total internal reflection (ATR) element, transparent in the infrared and whose surfaces were activated in view of covalently binding a receptor. Silicon and germanium ATR elements were considered. The original method is based on the grafting of bifunctional spacer molecules directly at the surface of the germanium crystal, avoiding the deposition of an intermediate metal layer. The grafting of these binding molecules (under their N-hydroxysuccinimidyl ester forms) was performed either by wet chemistry or by photochemistry. The functionalized surfaces, which allow the binding of molecules bearing peripherical NH2 groups, were successfully used, e.g., for the detection of proteins (streptavidin) or of small molecules (biotin). In the latter case, the biotin was readily detected for concentrations as low as 10(-12) M.