Rm. Wadkins et al., CALIBRATION OF BIOSENSOR RESPONSE USING SIMULTANEOUS EVANESCENT-WAVE EXCITATION OF CYANINE-LABELED CAPTURE ANTIBODIES AND ANTIGENS, Analytical biochemistry, 232(1), 1995, pp. 73-78
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.