Fluorescence detection in hydrodynamically focused sample streams: Reduction of diffusional defocusing by association of analyte with high-molecular-weight species

Fluorescence methods have been developed for ultra-low-level detection down to the single molecule level, In ultra-low-level analysis, one successful approach to background reduction is to use hydrodynamically focused sample streams coupled with focused laser excitation to reduce detection volumes. However, low-molecular-weight analytes such as fluorescent dyes or fluorescently tagged nucleotides can diffuse radially out of the sample stream before detection, which can cause a significant reduction in the detection efficiency, Diffusional spreading can cause sample stream mixing in multiplexed analyses, Also, axial diffusion can cause misordering of analyte molecules in DNA sequencing at high analysis rates. A new method is presented for reducing diffusion effects by incorporating a high-molecular-weight species that has a strong binding affinity for the analyte (anchor) into the sheath fluid. As the analyte starts to diffuse out of the sample stream, it associates with the anchor and effectively assumes the anchor's molecular weight, This association reduces the analyte's mobility to that of the anchor. Consequently, both radial and axial diffusion are reduced. Further, in favorable cases, this adduct can have significantly increased luminescence yields and photostability compared to the free analyte, Examples of several dye polymer combinations involving electrostatic and other binding modes are given that show the very beneficial effects of this approach. Extension to other systems is described.