Luminescent conjugated polymers (LCPs) have been frequently utilized for optical biosensors. The detection schemes of
these sensors are employing the light harvesting properties or the conformation sensitive optical properties of the
conjugated polymers. LCPs have been utilized as colorimetric and fluorescent sensing elements for the recording of
biological processes. However, LCPs have several limitations for being used as real time in vivo imaging agents. In this
regard, novel thiophene based molecular scaffolds, denoted luminescent conjugated oligothiophenes (LCOs) have been
developed. These LCOs are chemically defined molecules having distinct side chain functionalizations and a precise
number of thiophene units. Herein the utilization of LCOs as specific ligands for the pathological hallmarks underlying
protein misfolding diseases, such as Alzheimer's disease, is described. The use of the conformation sensitive optical
properties of the LCOs for spectral separation of these pathological entities in a diversity of in vitro, ex vivo or in vivo
systems is demonstrated. The protein aggregates are easily identified due to the conformation-dependent emission profile
from the LCOs and spectral assignment of protein aggregates can be obtained. Overall, these probes will offer practical
research tools for studying protein misfolding diseases and facilitate the study of the molecular mechanism underlying
Protein based infections such as prion diseases have lately attracted a large amount of interest, primarily due to the Mad Cow Epidemic in Great Britain, and the increase of Alzheimer's disease and related diseases in the ageing Western society. Infective proteins are very stable and almost untraceable prior to infection making them ideal as biological weapons. Particularly if the used agent is of human origin, the immunoresponse can be avoided, leaving no trace of the infectious agent. The transient nature of infectious oligomeric intermediates of misfolded proteins or peptide fragments that later matures into fibrillar aggregates makes them hard to study, and methods to detect and study these species are sparse. There exist a number of fluorescent probes that bind specifically to protein amyloidic structures. Thioflavins (ThT, ThS), Congo and Nile red, 4-(dicyanovinyl)-julolidine (DCVJ), as well as derivatives amino-8-naphtalene sulphonate (ANS, Bis-ANS) which are known to bind to the fibrillar or pre-fibrillar states with dissociation constants of typically 1 - 20 μM. Here, transthyretin (TTR), insulin and lysozyme were used as model proteins to detect different amyloid precursor states for diseases such as senile systemic amyloidosis, familial amyloidotic polyneuropathy (FAP) and iatrogenic amyloidosis. Specifically, the probes were employed in static assays to characterize protofibrillar and mature amyloid fibrillar states using steady state and time-resolved fluorescence techniques. Particularly, we investigate and report on the possibility to detect protofibrillar states at low concentration levels using modern fluorescence array detector systems in conjunction with lasers operating in the blue or ultraviolett wavelengths as excitation source. Results of ANS, ThT and a ThT analogue (abbreviated ThC) are discussed.
We report on the initial time-resolved luminescense and nonlinear absorption properties of two polythiophenes 3-substituted with chiral charged amino acid-derivatized substituents, POWT and POMT. The photo-physical characterization yielded quantum efficiency typically in the range 0.01 - 0.1, however, with two-photon absorption cross-section better than or similar to a typical two-photon reference chromophore, such as fluorescein. They were tested as conformational sensitive optical probes for the recording of pH-induced conformational changes of synthetic peptides, proteins and samples of protein amyloid fibrils characteristic of amyloid related diseases. Particularly, the POMT polyelectrolyte with the L-enantiomeric side chains is shown to favor this induction of well defined structure as judged by the circular dichroic signal as well as a stronger enhancement of luminescense for the L-form over the D-form complex. Furthermore, time-resolved fluorescense and two-photon induced fluorescence both also showed a difference in the complexation with the D and L form. This shows that the multi-photon excitation path can be an efficient means for chiral recognition of biomolecular complexes. It is demonstrated how the conjugated polyelectrolyte L-POMT can be used to spectrally image the formation of amyloid fibrils of insulin using both one- and two-photon absorption based fluorescence imaging.
Optical parametric oscillator (OPO) and sum-frequency mixing (SFM) devices are useful tools for constructing ultraviolet (UV) laser sources for fluorescence spectroscopy. Here, a compact UV-laser sources based on frequency conversion of an actively Q-switched Nd:YAG laser is presented. The second harmonic generation from a Nd:YAG laser was utilized as pump radiation for a periodically poled KTiOPO<sub>4</sub> nanosecond optical parametric oscillator. The OPO-signal and the remaining pump were spatially mode-matched for Type I SFM in a β-barium borate (BBO) crystal and UV radiation at 293 nm could be generated. This corresponds to a conversion efficiency of 2% with respect to the 532 nm harmonic radiation. The wavelength region accessible with this UV source is useful for chemical and biological sensing. Excitation of tryptophan at 293 nm for detection of fluorescence emission in ovalbumin and transthyretin was demonstrated.