Fluorescence Reporting Based on FRET Between Conjugated Polyelectrolyte and Organic Dye for Biosensor Applications - Advanced Fluorescence Reporters in Chemistry and Biology - page 436

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Fig. 8 The emission spectra of CCP/ssDNA 8 -TR mixtures in water with [ssDNA 8 -TR] ¼ 2 10

8 M, and (FU) ¼ 2.1 10 7 M (excitation at 385 nm)

PL spectra at this saturation point are shown in Fig. 8 . The sensitized TR emission
from 19/ssDNA 8 -TR is 2.3-fold more intense than that for 18/ssDNA 8 -TR, and is 3.6-
fold more intense than that for 17/ssDNA 8 -TR. Detailed study of the TR fluorescence
quenching upon ssDNA 8 -TR/CCP complex formation reveals that the fluorescence
quenching of TR emission is the lowest for ssDNA 8 -TR/19. The reduced TR fluores-
cence quenching originates from the decreased local dye concentration within the
19/ssDNA 8 -TR complexes. These data highlight that reduction of the CCP charge
density can lead to increased CCP-sensitized C* emission mainly as a result of the
reduced C* fluorescence quenching within the CCP/DNA-C* complexes.
According to the above examples, to optimize FRET between CCPs and dye-
labeled nucleic acid from the viewpoint of molecular structure, it is first necessary
to examine the donor/acceptor energy levels and the fluorescence behaviors of the
donor or the acceptor within the CCP/PNA-C* or DNA-C* complexes in addition
to spectral overlap, orientation and distance between the donor and the acceptor.
Mismatched energy levels can lead to PET that competes with the FRET process,
leading to low or even neglectable CCP-sensitized dye emission. The probability of
PET process can be minimized by the introduction of functional groups on the
polymer backbone to fine-tune the HOMO and LUMO energy levels of the donor or
the incorporation of molecular spacer to slightly increase the donor-acceptor
distance. With the same electronic backbone structure, further improvement in
CCP-sensitized dye emission can be accomplished by reducing the charge density
of CCPs, which mitigates the acceptor quenching within CCP/DNA complexes.
In addition to optimization of molecular structures, small variations in the
assay operation conditions can also induce similar effects for improved polymer-
sensitized dye emission [ 40 ]. For instance, introduction of organic solvent into
buffer can discourage PET owing to decreased dielectric constant of the solvent