Fluorescence Reporting Based on FRET Between Conjugated Polyelectrolyte and Organic Dye for Biosensor Applications - Advanced Fluorescence Reporters in Chemistry and Biology - page 429

possible situations upon excitation of the polymer donor. Situation A refers to the
ideal situation for FRET, where the highest occupied molecular orbital (HOMO)
and lowest unoccupied molecular orbital (LUMO) energy levels of the acceptor
are well contained within the orbital energy levels of the donor. Upon excitation
of the donor, FRET to the acceptor occurs to induce an emissive process. More-
over, direct excitation of the acceptor under situation A does not quench the
acceptor emission. This character is particularly useful for monitoring whether
PET process exists in the system and how strong it could be. On the contrary,
situation B favors PET, where both the electron affinity and the ionization
potential are higher in one of the optical partners [ 68 ].AsshowninScheme 7 ,
excitation of the donor would lead to PET to the acceptor, while excitation of the
acceptor would result in a similar charge-separated state via hole transfer to the
donor. Although Scheme 7 is widely used for choosing suitable optical partners
for a specific application, it becomes less accurate for intermediate cases because
of the neglect of the contributions from the exciton binding energy, the intermo-
lecular charge transfer state energy, and the stabilization of the charged species by
the medium [ 69 ]. The mechanism for the competition between FRET and PET is
complex and less understood for the CCP/C* pairs, but it is still probable to
minimize the PET process by molecular design of CCP and the careful choice of
donor/acceptor pairs.
Concomitant with these progresses in CCP-based DNA assays, it is gradually
recognized that interactions within the CCP/DNA complexes are crucial for signal
amplification [ 70 ]. Electrostatic attraction between positively charged CCP and
negatively charged phosphate groups in DNA/PNA-C* or DNA/DNA-C* plays a
vital role in bringing CCP and C* into close proximity to meet the FRET distance
requirement. Different from ideal FRET systems, where the distance between the
donor and the acceptor is fixed, the distance between CCP and C* varies because of
the dynamic complex structures of CCP/DNA/PNA-C* (or DNA-C*). Thus, FRET
between CCP and C* is much more complicated than ideal FRET process. Within
the macromolecular complexes, there are cross-interaction between CCP and C*
and self-interaction among C* or CCPs. Close association between CCP and DNA/
PNA-C * or DNA/DNA-C* favors FRET; whereas, self-interaction among CCPs
leads to polymer aggregation, which reduces the polymer quantum yield in solu-
tion. Self-interaction among C* causes dye fluorescence quenching, ultimately
leading to reduced signal amplification.
According to this understanding, it is possible to optimize the FRET conditions
to achieve high signaling emission, such as to increase the donor quantum yield
( Q D ), to improve the spectral overlap ( J ( )), to optimize the molecular orientation

( k ), and to shorten the distance between donor/acceptor pairs ( r DA ). In addition, it is
also important to select donor-acceptor pairs with well-matched energy levels to
minimize PET, and to develop strategies to solve the fluorescence self-quenching of
both CCP and C* upon CCP/DNA-C* complexation. However, it is noteworthy to
mention that the fluorescence quenching of CCP may not always have negative
impact on CCP-sensitized dye emission [ 71 ].

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