Structure, Emissive Properties, and Reporting Abilities of Conjugated Polymers - Advanced Fluorescence Reporters in Chemistry and Biology - page 376

aggregation of the polymer during turn-on assays can confuse the signal if the
chains begin to self-quench, lowering the emission, while interaction with the target
simultaneously raises it.

5.2.1 “Turn-On” From Quencher Displacement

The flip side of the super-quenching phenomenon seen with conjugated polymer
molecular wires is that to use these polymers in a sensing scheme where a quench-
ing species is removed as part of target identification has the disadvantage that all
the binding sites along the exciton path have to be vacated or otherwise altered for
the polymer to become fluorescent. This requirement points to using polymers, such
as PPV, which have shorter conjugation lengths. Conversely, another problem with
this approach is background emission from insufficiently quenched polymer seg-
ments. This has been addressed by pretreating polymers with effective quenchers,
such as Cu(II), to reduce initial emission. The quenchers are removed during the
assay; this approach was the basis for sensitive and selective detection of Fe 2+ [ 93 ].
Quencher displacement has also been used for the detection of biological targets.
One approach is for the quencher to be removed by an entity that competitively
binds to it. For example, Whitten and co-workers developed a system where the
biotin-avidin interaction was used to remove a biotin-MV 2+ quencher from sulfo-
nated PPV [ 12 ]. Subsequent studies by Bazan and co-workers on similar systems,
however, showed that nonspecific protein interactions with the polyelectrolyte also
led to increases in the emission of the quenched complex. In a buffer that promotes
avidin-biotin binding, addition of avidin actually increased quenching. They attrib-
uted this to the avidin-biotin-quencher complex associating more strongly with the
polymer in buffer than the biotin-quencher alone [ 94 ]. In another model study,
Heeger and co-workers demonstrated using antibodies to remove quenchers from a
sulfonated PPV complexed with a cationic (nonconjugated) polyelectrolyte system
- the cationic polymer reduced the nonspecific interactions with the PPV [ 54 ].
Interestingly, both positive and negative quenchers interacted with the PPV/cat-
ionic polymer complex quenching the PPV emission. Addition of antibodies spe-
cific to the quenchers led to increase in the PPV emission. In a demonstration of
detection of a more interesting target, Whitten and co-workers preloaded sulfonated
PPV with Cu 2+ to reduce emission then added a oligohistidine-tagged anti-hepatitis
C antibody to form a complex with Cu 2+ , which quenched the emission further [ 95 ].
Addition of hepatitis C antigen led to the restoration of the emission. Quencher
displacement has also been used to detect protease activity [ 7 , 86 ] using conjugated
polyelectrolytes and peptide substrates of opposite charge linked to quenchers.
Cleavage of the substrate leads to the disassociation of the (neutral) quencher,
which then diffuses away, turning on the emission.
A related type of turn-on assay has also been developed using competition
between a quencher and a target for binding sites associated with the emissive
polymer. The more target presented, the less the quenchers can interact with the
polymer - this registers as a rise in emission. An illustration of how a turn-off