Page 368 - Organic Electronics in Sensors and Biotechnology

Luminescent Conjugated Polymers for Staining and Characterization of Amyloid Deposits 345

diseases are associated with conformational changes in proteins.
Methods for the detection and quantification of diverse aggregated
states of proteins are also of great importance with respect to the
long-term stability and production of peptide pharmaceuticals in
commercial pharmaceutical formulations used for the treatment of
various diseases, e.g., insulin for diabetes. Although, it is rare to observe
iatrogenic (i.e., treatment-induced) protein aggregates due to admin-
istration of peptide pharmaceuticals, the therapeutic effect of the
peptide drug becomes limited if the peptide has been converted into
amyloid fibrils.
LCPs, with their unique structural related optical properties, have
proved to be an exceptionally powerful tool to study the amyloid
fibrillation event. Novel conformation-sensitive optical methods for
the detection of formation of amyloid fibrils in bovine insulin and
chicken lysozyme based on conformational changes of the anionic
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polythiophene derivative, PTAA, were recently reported. The tech-
nique is based on noncovalent assembly of the LCP and the different
forms of the proteins (Fig. 9.7a). Depending on the conformation of the
protein, different emission spectra from PTAA are observed (Fig. 9.7b).
Upon binding to the native monomeric form of insulin, PTAA emits
light with emission maximum of 550 nm, whereas PTAA bound to
amyloid fibrils of insulin is emitting light with lower intensity and
the emission maximum is red-shifted to 580 nm. The red-shift of the
emission maximum and the decrease of the emission intensity from
PTAA are associated with a planarization of the polymer backbone
and an aggregation of adjacent polymer chains. Hence, specific opti-
cal fingerprint is achieved for the β-sheet containing amyloid fibrils.
The detection of insulin fibrils can also be observed by absorption
and visual inspection, and this can be useful for the development of
simple screening methods for the detection of amyloid fibrils. Fur-
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thermore, when plotting the ratio of emitted light at 550 nm and
580 nm, ratio 550/580 nm, the formation of insulin fibrils can be mon-
itored (Fig. 9.7c). The kinetic plot is showing a lag phase, followed by
a growth phase and a plateau phase, which are characteristic for the
formation of amyloid fibrils.
PTAA binds both the native form of insulin and the fibrillar form
of insulin. Nevertheless, these forms can be easily distinguished, due
to the conformational changes of the polymer backbone upon bind-
ing to the different forms of the proteins, as minor perturbations of
the geometry of the polymer backbone can be reflected as alterations
of the electronic structure of the conjugated backbone. Thus, binding
of the polymer to different forms of proteins will give rise to different
optical features for the LCP. This is an improvement over small amy-
loid ligands, such as Congo red or thioflavins, as these probes only
change in optical feature whether they are free in solution or binding
to pockets in the protein or to the surface of the protein. So far, it has
been shown that LCPs can be used to distinguish between the native