Post-column reaction detection based on fluorescence energy transfer in the far red spectral region

Post-column reaction detection may result in enhanced analytical sensitivity and selectivity. This paper describes an on-line HPLC with post-column fluorescence energy transfer assay using biotin as a model analyte. Biotin labeled with R-phycoerythrin was used as the donor labeled ligand and streptavidin labeled with an indodicarbocyanine dye (Cy5), the acceptor labeled binder protein. The use of these labels provided a detection wavelength in the far red spectral region which is more selective for biological samples. In the on-line system, biotin was injected into the HPLC system followed by Cy5 labeled streptavidin and R-phycoerythrin labeled biotin, post-column. The mixture was incubated on-line in an open tubular reactor coil maintained at 37 degrees C. The measured response was the sensitized emission of Cy5 due to fluorescence energy transfer from R-phycoerythrin labeled biotin measured at 670 nm. Excitation was at 488 nm, which provided a large Stokes shift for reduction of scatter interference. The system was optimized with regard to the post-column reagents to obtain the minimum detectable concentration while maintaining appropriate dynamic range for the analysis of biotin. Biotin spiked in 0.01 M phosphate buffer, pH 7.4, showed a dynamic range of 304.0 pg/ml-122.20 ng/ml with a correlation coefficient of 0.993. The limit of detection for this assay was 304.0 pg/ml. The precision calculated at the blank (n = 6) was 4.14%.