InP-Bovine Serum Albumin Conjugates as Energy Transfer Probes.

The use of indium phosphide (InP) quantum dots (QDs) as biological fluorophores is limited by the low photoluminescence quantum yield (ϕPL) and the lack of effective bioconjugation strategies. The former issue has been addressed by introducing a strain relaxing intermediate shell such as ZnSe, GaP etc. that significantly enhances the ϕPL of InP. Herein, we present an effective strategy for the conjugation of emissive InP/GaP/ZnS QDs with a commonly used globular protein, namely bovine serum albumin (BSA), which generate colloidally stable QD bioconjugates, labeled as InP-BSA and demonstrate its use as energy transfer probes. The conjugate contains one protein per QD, and the circular dichroism spectra of BSA and InP-BSA exhibit similar fractions of α-helix and ß-sheet, reflective of the fact that the secondary structure of the protein is intact on binding. More importantly, the fluorescence polarization studies corroborate the fact that the bound protein can hold a variety of chromophoric acceptors. Upon selectively exciting the InP-BSA component in the presence of bound chromophores, a reduction in the emission intensity of the donor is observed with a concomitant increase in emission of the acceptor. Time-resolved investigations further confirm an efficient nonradiative energy transfer from InP-BSA to the bound acceptors.

Fluorescent ratiometric MFC probe sensitive to early stages of alpha-synuclein aggregation.

We introduce a sensor molecule, AS140-MFC, consisting of a covalent adduct of an Ala-to-Cys mutant of alpha-synuclein with the 3-hydroxychromone dual emission dye MFC. We show that the AS140-MFC construct is a multiparametric fluorescent probe suitable for the continuous monitoring of protein aggregation and is sensitive to the early and intermediate stages of alpha-synuclein aggregation, a process associated with Parkinson's disease.

Multiparametric fluorescence detection of early stages in the amyloid protein aggregation of pyrene-labeled alpha-synuclein.

The aggregation of alpha-synuclein, a presynaptic protein, has an important role in the etiology of Parkinson's disease. Oligomers or protofibrils adopting the cross-beta-sheet structure characteristic of fibrillating amyloid proteins are presumed to be the primary cytotoxic species. Current techniques for monitoring the kinetics of alpha-synuclein aggregation based on fluorescent dyes such as Thioflavin-T and Congo red detect only the terminal fibrillar species, are discontinuous and notoriously irreproducible. We have devised a new fluorescence aggregation assay that is continuous and provides a large set of fluorescence parameters sensitive to the presence of oligomeric intermediates as well as fibrils. The approach involves tagging functionally neutral Ala-to-Cys variants of alpha-synuclein with the long-lifetime fluorophore pyrene. Upon induction of aggregation at 37 degrees C, the entire family of steady-state descriptors of pyrene emission (monomer intensity, solvent polarity ratio (I(I)/I(III)), and anisotropy; and excimer intensity) change dramatically, particularly during the early stages in which oligomeric intermediates form and evolve. The pyrene probe senses a progressive decrease in polarity, an increase in molecular mass and close intermolecular association in a manner dependent on position in the sequence and the presence of point mutations. The time-resolved decays (0-160 ns) of intensity and anisotropy exhibited complex, characteristic features. The new assay constitutes a convenient platform for the high-throughput screening of agents useful in the diagnosis and therapy of Parkinson's disease as well as in basic investigations.