FLUORESCENT AND PHOSPHORESCENT STUDY OF LANGMUIR-BLODGETT ANTIBODY FILMS FOR APPLICATION TO OPTICAL IMMUNOSENSORS

摘要

The application of Langmuir-Blodgett films of antibodies as sensitive elements of biosensors based on the TIRF and plasmon resonance principles requires data on the regularity of the monolayers and activity of the antibodies forming these monolayers. The monolayers of the monoclonal anti-Pd coproporphyrin antibodies, carrying a europium label, were formed on water/air boundary by the Langmuir-Blodgett method. Films were then deposited onto the surface of a plate, modified with a thin layer of different siloxane polymer carriers for covalent attachment of the protein monolayer. We used a methodological approach for protein films investigation that consists in monolayer parameters evaluation by means of registration of europium-labeled antibodies fluorescence, that form the film. Under surface pressure values the 30-35 mN/m (the area for IgG molecule in the film being 6000-4000 A) film takes on tight packing peculiar for Langmuir-Blodgett films. The distribution coefficient between the water-air interface and the subphase for the antibodies at different surface pressures, temperatures and pH values was measured. The range for the distribution coefficient is 0.02-0.01 for IgG molecules. The immunological activity of IgG molecules and steric hindrances for antigen binding in the LB films were estimated by measurement of the intensity of the phosphorescence of Pd-coproporphyrin conjugated with proteins of various molecular weight. The LB films of monoclonal antibodies to Pd-coproporphyrin retained 90% immunological activity for Pd-coproporphyrin (MW 760) binding, 17% binding of Pd-coproporphyrin conjugated with SIT (soybean inhibitor of trypsin, MW 25 000) and 0.44% binding of Pd-coproporphyrin conjugated with TG (thyroglobulin, MW 669 000).