Structure, Emissive Properties, and Reporting Abilities of Conjugated Polymers - Advanced Fluorescence Reporters in Chemistry and Biology - page 373

systems with only 3-5 species. Sample processing steps can be used to remove
interferents and/or concentrate target species; however, addition of these steps
complicates sensor device design.

5.1.3 “Turn-Off” Detection of Biological Targets

Turn-off sensing has also been used for the detection of biological targets: enzymes,
other proteins, and DNA hybridization. Conjugated polyelectrolyte turn-off sensing
has been used for the detection of DNA hybridization and the identification of
complementary strands through direct [ 82 ] and indirect bead-based assays [ 83 ]
though these materials lack the exceptional sensitivity shown by PT DNA hybridi-
zation sensors developed by Leclerc and colleagues [ 84 , 85 ] and discussed in
Sect. 5.2.2 .
Sensing schemes for detecting enzymes depend on using the enzymatic action -
bond cleavage or functional group modification - to create a quencher. In one
example of enzymatic sensing, rhodamine-110 diarginine amide was mixed with
PPE with carboxylate side chains and nanomolar papain added [ 86 ]. The protease
cleaved one arginine amide, creating the positively charged Rhodamine-Arg mo-
lecule that associated electrostatically with the negative PPE and quenched its
emission through singlet-singlet energy transfer. A similar approach from Whitten
and co-workers deployed beads coated with polyelectrolyte conjugated polymers
and avidin proteins combined with a biotin-peptide-quencher [ 87 ]. Reduction in
quenching signaled protease activity as cleavage of the peptide by proteases
severed the connection between the biotin and the quencher and prevented quench-
ing of the polymer upon the biotin binding to the avidin (Fig. 10 ). Interestingly, it
was necessary to carry out the peptide/protease reactions before adding the beads -
the enzymes were not able to cleave bead-bound peptide sequences. Kinase detec-
tion via quenching was achieved with PPE/carboxylate side chains on a bead loaded
with Ga 3+ ions [ 88 ]. The beads were mixed with a kinase substrate labeled with
rhodamine; when the kinase phosphorylated the peptide the PO 3 groups bound to
the Ga 3+ ions, bringing the quencher into proximity of the polymer and quenching
its emission.
Nonenzymatic proteins have also been detected with turn-off conjugated polymer
sensors. Unlike enzymatic assays, these assays do not depend on the protein having
an action on a substrate. For redox-active proteins, the proteins themselves can act as
the quenching agent; for example, Heeger and co-workers showed that sulfonated
PPV can be quenched by cytochrome C (cyt C) [ 89 ]. Interestingly, a later study by
Waldeck and co-workers, using anionic PPP mixed with cyt C, myoglobin and
polyamidoamine dendrimer, showed that quenching does not require that the protein
(or protein analog) engage in electron transfer but can occur because of conforma-
tional changes caused by electrostatic-driven aggregation. Bunz and co-workers have
studied the nonspecific interactions of positive, neutral, and negative proteins with
PPEs containing carboxylate sidechains and their effects on emission [ 90 ]. The
different proteins studied quenched the polymer emission by varying combinations