Waveguide multi-channel immunoassay using photo-deprotection immobilization

A planar optical waveguide was used to simultaneously excite fluorescence due to antigen binding in three separate areas of immobilized antibody. Biotin labeled, polyclonal antibodies to goat, human, and rabbit IgG were immobilized through surface bound, photo-activated MeNPOC-biotin-bSA and avidin. Exposing the MeNPOC to UV light effectively uncaged the biotin molecule attached to the bSA and allowed avidin, followed by the biotin labeled antibody, to bind to the waveguide surface. Whereas a time intensive, non-specific binding prone step-and-repeat method is normally used to form the individual capture layers, we chose to pursue a combined deposition method involving sample wells (to separate the different capture antibodies) and photo-activated crosslinkers (to bind antibodies to the surface). The result was a covalently linked multi-component capture layer formed in a short period of the time. Specific and cross-reactive activities of this antibody array were gauged by sequentially injecting analyte specific to one antibody area at a time. Results suggested that the binding of each analyte occurred predominately in the correct area and, depending on the particular antibody, generated varying levels of cross reactivity. A comparison of results with previously acquired, physically capture layer data did not infer one deposition technique was better than the other.

Duke Scholars

Author William M. Reichert Biomedical Engineering