PHOTOAFFINITY IMMOBILIZATION OF FUSION PROTEINS ON BIOSENSOR SURFACES

THIS PROJECT IS DESIGNED TO RESOLVE A CRITICAL AND ENDURING OBSTACLE TO THE DEVELOPMENT OF DEPENDABLE BIOSENOR SYSTEMS FOR THE TIMELY DETECTION OF TOXIC AND INFECTIOUS AGENTS. A PASSIVATED SENSOR SURFACE WILL BE DEVELOPED THAT WILL ASSOCIATE UNIQUELY WITH THE SPECIFIC BINDING PROTEIN IN ITS CRUDE SOURCE SOLUTION AND, UPON ACTIVATION BY LIGHT, COVALENTLY BOND THIS PROTEIN FUNCTIONALLY ORIENTED IN A STABLIZING, BIOMOLECULE-COMPATIBLE ENVIRONMENT. A TRIFUNCTIONAL AMINO ACID WILL BE DERIVATIZED ON ONE SITE WITH A PHOTOACTIVATABLE REAGENT CAPABLE UPON ILLUMINATION OF FORMING COVALENT BONDS WITH ESSENTIALLY ANY ORGANIC TARGET GROUP (INCLUDING ALL PROTEINS) UNDER MILD REACTION CONDITIONS. THIS REAGENT WILL THEN BE THERMOCHEMICALLY BONDED TO A LIGAND FOR THE SPECIFIC BINDING PROTEIN AND TO THE SENSOR SURFACE PREVIOUSLY PASSIVATED WITH A PHOTOREACTIVE HYDROGEL, TO PROVIDE AN ESSENTIALLY AQUEOUS ENVIRONMENT CONTAINING CHOSEN LIGAND AND PHOTOREACTIVE GROUPS ADJACENT ON THE SENSOR SURFACE. THIS PASSIVATED PHOTOAFFINITY SENSOR SURFACE WILL BE EXPOSED TO AQUEOUS PROTEIN MIXTURES AND TESTED FOR ITS EFFICIENCY AND SPECIFICITY IN PHOTOCHEMICAL IMMOBILIZATION OF THE DESIRED BINDING PROTEIN, AS WELL AS THE STORAGE AND USE STABILITY OF THE IMMOBILIZED SENSOR PROTEIN. IN THE PHASE I EFFORT A PASSIVATING COATING FOR GLASS SURFACES AND A PHOTOREACTIVE TRIFUNCTIONAL REAGENT WILL BE PREPARED, WHICH WILL BE REACTED WITH THE PASSIVATED SURFACE AND WITH A LIGAND TO PROVIDE A PHOTACTIVATIBLE AFFINITY SURFACE. THIS PHOTOAFFINITY GLASS IS EXPECTED TO REDUCE NON-SPECIFIC PROTEIN BINDING BY AT LEAST 75% AND TO IMMOBOLIZE THE FUSION PROTEIN WITH GREATER THAN 50% RECOVERY OF DNA-BINDING ACTIVITY. PHASE II WILL OPTIMIZE THE REAGENTS AND COATING PROCESS FOR TWO OR MORE FUSION PROTEINS AND DEMONSTRATE THE FEASIBILITY OF THIS CHEMISTRY FOR PATTERNED IMMOBILIZATION ON BIOSENSOR SURFACES.