Circularly Polarized Luminescence of [6]Helicenes through Excited-State Intramolecular Proton Transfer

We present the concept of combining circularly polarized luminescence (CPL) and excited-state intramolecular proton transfer (ESIPT) features into a single molecule as a strategy to generate high-performance ESIPT-based CPL materials. For this purpose, a [6]helicene bearing two ESIPT structural units was synthesized using a double Suzuki–Miyaura reaction and a double C(sp2)−H hydroxylation approach. The photophysical properties of the doubly hydroxylated [6]helicene were studied in parallel with a non-hydroxylated [6]helicene control compound, revealing that the presence of a chiral [6]helicene unit results in a strong CPL response and the presence of the ESIPT units in a considerable red shift. The red-shifted emission along with the outstanding glum (≈10−2) and a large Stokes shift makes the doubly hydroxylated [6]helicene a promising candidate for use in optoelectronics

Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies.This work is funded by grant P10-FQM-6154 from the Consejeria de Innovacion, Ciencia y Empresa (Junta de Andalucia)

Fluorescence Lifetime Imaging Microscopy for the Detection of Intracellular pH with Quantum Dot Nanosensors

While the use of quantum dot (QD) nanoparticles for bioimaging and sensing has been improved and exploited during the last several years, most studies have used emission intensity-based techniques. Fluorescence lifetime imaging microscopy (FLIM) can also be employed for sensing purposes, overcoming many of the limitations of the aforementioned systems. Herein, we show that the photoluminescence (PL) lifetime of mercaptopropionic acid-capped QDs (MPA-QDs) collected from FLIM images can be used to determine intracellular pH. The PL average lifetime of MPA-QDs varied from 8.7 ns (pH < 5) to 15.4 ns (pH > 8) in media mimicking the intracellular environment. These long decay times of QD nanoparticles make them easily distinguishable from intrinsic cell autofluorescence, improving selectivity in sensing applications. We demonstrate, for the first time, the successful detection of changes in the intracellular pH of different cell types by examining the PL decay time of QDs. In particular, the combination of FLIM methodologies with QD nanoparticles exhibits greatly improved sensitivity compared with other fluorescent dyes for pH imaging. A detailed description of the advantages of the FLIM technique is presented

Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

Abstract: Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies. Int. J. Mol. Sci. 2012, 13 940

Similarity between the kinetic parameters of the buffer-mediated proton exchange reaction of a xanthenic derivative in its ground- and excited-state

Buffer-mediated proton exchange reactions of a xanthenic dye were studied in the ground and the excited state by single molecule and bulk fluorescence techniques, respectively. The rate constant obtained supported the uniformity of the process in the ground and the excited state, and the need of adequate character of the buffer species to be able to promote excited-state reactions

Two-Step Amyloid Aggregation: Sequential Lag Phase Intermediates.

The self-assembly of proteins into fibrillar structures called amyloid fibrils underlies the onset and symptoms of neurodegenerative diseases, such as Alzheimer's and Parkinson's. However, the molecular basis and mechanism of amyloid aggregation are not completely understood. For many amyloidogenic proteins, certain oligomeric intermediates that form in the early aggregation phase appear to be the principal cause of cellular toxicity. Recent computational studies have suggested the importance of nonspecific interactions for the initiation of the oligomerization process prior to the structural conversion steps and template seeding, particularly at low protein concentrations. Here, using advanced single-molecule fluorescence spectroscopy and imaging of a model SH3 domain, we obtained direct evidence that nonspecific aggregates are required in a two-step nucleation mechanism of amyloid aggregation. We identified three different oligomeric types according to their sizes and compactness and performed a full mechanistic study that revealed a mandatory rate-limiting conformational conversion step. We also identified the most cytotoxic species, which may be possible targets for inhibiting and preventing amyloid aggregation

Bulk and Single-Molecule Fluorescence Studies of the Saturation of the DNA Double Helix Using YOYO‑3 Intercalator Dye

We report a thorough photophysical characterization of the interactions between double-stranded DNA (dsDNA) and the trimethine cyanine homodimer dye YOYO-3. The fluorescence emission of this dye is enhanced by intercalation within the DNA double helix. We have explored the saturation of the dsDNA by bound YOYO-3 at the single-molecule level by studying the single-pair Förster resonance energy transfer (FRET) from an energy donor, Alexa Fluor 488, tagged at the 5′ end of the double helix and the energy acceptor, YOYO-3, bound to the same DNA molecule. The spontaneous binding of YOYO-3 gives rise to an effective distribution of different FRET efficiencies and, therefore, donor–acceptor (D–A) distances. These distributions reveal the existence of multiple states of YOYO-3. Steady-state and time-resolved fluorescence and circular dichroism confirmed the presence of a DNA-bound aggregate of YOYO-3, conspicuous at high dye/base pair ratios. The spectral features of the aggregate suggest that it may have the structure of a parallel H-aggregate

Interaction of YOYO‑3 with Different DNA Templates to Form H‑Aggregates

Homodimeric cyanine dyes are DNA intercalators that display a large enhancement of fluorescence emission when bound to double-stranded DNA. However, other different interaction modes are possible, such as H-type molecular aggregates of the dye, templated by the nucleic acid. In this paper, we study in depth the formation of nonfluorescent H-aggregates of the cyanine homodimer YOYO-3 with two different DNA templates using absorption and both steady-state and time-resolved fluorescence spectroscopy. First, a nonfluorescent YOYO-3 H-aggregate complex was found to form in single-stranded polycytidine chains, resulting in the appearance of a new absorption band at approximately 500 nm. The specific interaction of cytosine bases suggests the involvement of the C-rich i-motif in facilitating the formation of the H-aggregate complex. Second, the interaction of YOYO-3 with double-stranded poly­(A·T) tracts also led to the appearance of a new absorption band at approximately 500 nm, and hence of a different type of H-aggregate. We found that the aggregate is formed mainly in double-stranded regions with consecutive adenine bases in the same strand (and thymine bases in the complementary strand). These poly­(A·T) tracts provide narrow minor grooves and enhanced electrostatic negative potential to promote the aggregation of the negatively charged cyanine. As the YOYO-3 H-aggregates are nonfluorescent, our results provide an important basis to quantitatively understand the fluorescence emission of this cyanine dye in the presence of DNA strands