FRET and competing processes between conjugated polymer and dye substituted DNA strands: a comparative study of probe selection in DNA detection

Hameed A Al Attar, Andy P Monkman
Biomacromolecules 2009 May 11, 10 (5): 1077-83
Fluorescence resonance energy transfer (FRET) between water-soluble conjugated polymer, poly-(9,9-bis(6'-N,N,N-trimethylammonium)-hexyl-fluorene phenylene) bromide, and ssDNA's labeled with four different types of dyes (Pacific-blue, Alexa-fluor 430, Fluorescein, and ROX) has been investigated. The effect of spectral overlap and Stokes-shift on the efficiency and properties of FRET were studied. In the DNA sequence detection technique that using cationic conductive polymer and the negatively charged DNA the electrostatic interaction leads to strong aggregation. The effective concentration of these aggregates is quite high leading to strong self-absorption. In this case, labeling with small Stokes shift dyes shows a strong output emission limitation even in extremely dilute system. The steady state fluorescence quenching of the CCP by FRET reveals that the competition between FRET and self-absorption plays a major role when accounting for the FRET ratio. Time-resolved fluorescence lifetime analysis was carried out to measure the energy transfer between the donor and the acceptor excluding self-absorption and quenching by ground-state complex formation. Time-resolved analysis indicates only around 30% of the total CCP excited-state population is quenched by FRET, whereas 55% is quenched by the DNA/buffer solution.

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