Simultaneous measurement of Cy5.5-labeled capture antibodies and Cy5-labeled antigens in a fiber optic biosensor: photobleaching and resonance energy transfer effects during fiber regeneration

Fiber optic biosensors using evanescent wave excitation of fluorescence have proven their ability to detect antigens rapidly in a variety of environmental and clinical samples. One problem associated with these biosensors is the fiber-to-fiber variability in measured signal. We have addressed this problem by labeling an immobilized anti-trinitrotoluene ((alpha) TNT) capture antibody with the fluorescent cyanine derivative Cy5.5 (emission (lambda) max equals 696 nm). The antigen (a TNT analog) was then labeled with fluorescent Cy5 (emission (lambda) max equals 668 nm). Both fluorophores were excited by 635 nm light, and their emission was collected using a fiber optic spectrometer. The fluorescence from the Cy5.5 labeled capture antibody served as a calibration signal for each fiber and was used to correct for differences in optics, fiber defects, and varying amounts of immobilized capture antibody. The calibration process could be used repeatedly following fiber regeneration. However, when each immobilized antibody was labeled with at least one Cy5.5 fluorophore, fluorescence resonance energy transfer (FRET) was observed between the Cy5-antigen donor and the Cy5.5-labeled acceptor. The extent of FRET affected the measured antigen and calibration signal, and these signals had to be adjusted accordingly. We describe the procedures to account for fluorescently labeled antibodies during extended biosensor use.