Covalent immobilization of protein monolayers for biosensor applications.

Various sensor technologies require a monolayer of tightly bound proteins. Five methods for protein immobilization were evaluated, comprising two methods of linker attachment to a silicon nitride surface (CNBr, silanization) and three methods of linker attachment to the protein (via primary amines, carboxyl groups, and the aromatic rings of tyrosine and histidine). These covalent binding methods were evaluated by immobilization of the enzyme alkaline phosphatase and with monoclonal antibodies tagged with 125I. It was determined that approximately 75% of the protein on the "covalently" immobilized samples was simply adsorbed. Adsorption was reduced by varying the pH and ionic strength of the wash buffers as well as by adding chaotropes and competitors to the wash buffer. A more efficient method of reducing adsorption was to perform the immobilization in the presence of a detergent; 0.5% Tween 60 was the most effective detergent studied. The optimized procedure gives a surface loading of 1.20 x 10(12) MAb/cm2. of which approximately 10% was adsorbed protein. This surface density is consistent with a monolayer of immobilized protein. This protein was shown to be active by challenging immobilized antibodies with 35S-labelled antigen; only the antibody/antigen pair released radioactive material upon elution.