Synthesis and fluorescence study of water-soluble conjugated polymers for efficient FRET-based DNA detection

Two cationic conjugated polyelectrolytes (CPs, P1i and P2i) were synthesized and examined as a fluorescence resonance energy transfer (FRET) donor to fluorescein (Fl)-labeled single-stranded DNA (ssDNA-Fl) using steady-state and time-resolved photoluminescence (PL) spectroscopy. The two polymers have the same π-conjugation with the main structural difference being the presence of the spiro-anthracenyl substituents orthogonal to the polymer backbone of P2i. These spiro-substituents can function as a molecular spacer that increases the intermolecular separation in the electrostatic complex with ssDNA-Fl. We measured almost complete PL quenching of the excited Fl* after electrostatic complexation with P1i (PL lifetime 4 ns → 78 ps) and relatively moderate quenching with P2i (PL lifetime 4 ns → 552 ps). A quenching efficiency (Φ_eT) of 98% and 86% was obtained for P1i/ssDNA-Fl and for P2i/ssDNA-Fl, respectively. Both systems have same thermodynamic driving force for quenching as a result of them having the same electronic structures. This discrepancy can be explained in terms of the reduced quenching (via electron transfer, eT) by the increased D-A distance due to the existence of spiro-attached molecular spacers in P2i. It shows that thermodynamically favorable eT quenching can be controlled kinetically by modulating the D-A intermolecular distance using molecular spacers, which suggests an important molecular design guideline for efficient CPs-based DNA detection.