CALIBRATION OF BIOSENSOR RESPONSE USING SIMULTANEOUS EVANESCENT-WAVE EXCITATION OF CYANINE-LABELED CAPTURE ANTIBODIES AND ANTIGENS

Fiber optic biosensors have proven their ability to detect antigens ra pidly in a variety of environmental and clinical samples. These biosen sors are based on the technique of covalently linking antibodies to th e core of an optical fiber and detecting antigen binding via measureme nt of fluorescence induced in the evanescent wave. One problem associa ted with these biosensors is the fiber-to fiber variability in measure d signal. We have addressed this problem by labeling a portion of the immobilized capture antibody with the fluorescent cyanine dye Cy5.5 (e mission lambda(max) = 696 nm). The antigen was then labeled with fluor escent Cy5 (emission lambda(max) = 668 nm). Both fluorophores were exc ited by 635-nm light, and their emission was collected using both a fi ber optic spectrometer and a biosensor optimized to collect fluorescen ce at two wavelengths. The fluorescence from the Cy5.5-labeled capture antibody served as a calibration signal for each fiber and corrected for differences in optics, fiber defects, and varying amounts of captu re antibody present on the fiber. Our data show that normalizing the s ignal measured from Cy5-labeled antigen binding to the Cy5.5 signal pr ovides a standardization process for greatly reducing signal variance among individual fibers. (C) 1995 Academic Press, Inc.