Three species multiplexing of fluorescent dyes and gold nanoclusters recovered with fluorescence lifetime correlation spectroscopy.

Biosensor applications often require the simultaneous detection of multiple analytes, with a clear need to go beyond the traditional multiplexing relying on distinct fluorescent dyes across the visible spectrum. Fluorescence lifetime correlation spectroscopy (FLCS) is a powerful approach taking advantage of the fluorescence lifetime information to separate the contributions of different fluorescent species with overlapping emission spectra. However, so far FLCS detection has been demonstrated only on binary mixtures of two fluorescent dyes, limiting its multiplexing capabilities. Here, we report the first quantitative FLCS measurements within a ternary mixture composed of three different fluorescent emitters with near-identical emission spectra. Two organic fluorescent dyes, Alexa Fluor 647 and CF640R, are combined with water-soluble Au18(SG)14 gold nanoclusters. Our experimental data establish that FLCS allows to accurately determine individual concentrations within intricate ternary mixtures. Another major aspect of interest concerns the assessment of the suitability of gold nanoclusters for FLCS multiplexing applications. With their microsecond lifetime and stable emission characteristics, gold nanoclusters add a valuable new aspect to the array of FLCS probes. Extending FLCS multiplexing beyond binary mixtures paves the way for further progress in the simultaneous highly parallel biosensing of multiple species.

Unveiling the photoluminescence dynamics of gold nanoclusters with fluorescence correlation spectroscopy.

Gold nanoclusters (AuNCs) have captured significant interest for their photoluminescent properties; however, their rapid photodynamics remain elusive while probed by ensemble-averaging spectroscopy techniques. To address this challenge, we use fluorescence correlation spectroscopy (FCS) to uncover the photoluminescence dynamics of colloidal Au18(SG)14 nanoclusters. Our FCS analysis reveals the photoluminescence (PL) brightness per nanocluster, elucidating the impact of photoexcitation saturation and ligand interactions. Unlike DNA-encapsulated silver nanoclusters, their gold counterparts notably exhibit minimal blinking, with moderate amplitudes and 200 µs characteristic times. Our data also clearly reveal the occurrence of photon antibunching in the PL emission, showcasing the quantum nature of the PL process, with each AuNC acting as an individual quantum source. Using zero-mode waveguide nanoapertures, we achieve a 16-fold enhancement of the PL brightness of individual AuNCs. This constitutes an important enabling proof-of-concept for tailoring emission properties through nanophotonics. Overall, our study bridges the gap between ensemble-averaged techniques and single-molecule spectroscopy, offering new insights into AuNC photodynamics for biosensing and imaging applications.

BF2-Functionalized Benzothiazole Amyloid Markers: Effect of Donor Substituents on One- and Two-Photon Properties.

Investigation of amyloids with the aid of fluorescence microscopy provides crucial insights into the development of numerous diseases associated with the formation of aggregates. Here, we present a series of BF2-functionalized benzothiazoles with electron-donating methoxy group(s), which are tested as amyloid fluorescent markers. We evaluate how the position of donor functional group(s) influences optical properties (fluorescence lifetime (τ) and fluorescence quantum yield (FQY)) in a solution and upon binding to amyloids. We elucidate the importance of surrounding environmental factors (hydrogen-bonding network, polarity, and viscosity) on the observed changes in FQY and evaluate how the localization of a donor influences radiative and nonradiative decay pathways. We conclude that a donor attached to the benzothiazole ring contributes to the increment of radiative decay pathways upon binding to amyloids (kr), while the donor attached to the flexible part of a molecule (with rotational freedom) contributes to a decrease in nonradiative decay pathways (knr). We find that the donor-acceptor-donor architecture allows us to obtain 58 times higher FQY of the dye upon binding to bovine insulin amyloids. Finally, we measure two-photon absorption (2PA) cross sections (σ2) of the dyes and their change upon binding by the two-photon excited fluorescence (2PEF) technique. Measurements reveal that dyes that exhibit the increase/decrease of σ2 values when transferred from highly polar solvents to CHCl3 present a similar behavior upon amyloid binding. Our 2PA experimental values are supported by quantum mechanics/molecular mechanics (QM/MM) simulations. Despite this trend, the values of σ2 are not the same, which points out the importance of two-photon absorption measurements of amyloid-dye complexes in order to understand the performance of 2P probes upon binding.

Zn(II) binding to pramlintide results in a structural kink, fibril formation and antifungal activity.

The antimicrobial properties of amylin, a 37-amino acid peptide hormone, co-secreted with insulin from the pancreas, are far less known than its antidiabetic function. We provide insight into the bioinorganic chemistry of amylin analogues, showing that the coordination of zinc(II) enhances the antifungal properties of pramlintide, a non-fibrillating therapeutic analogue of amylin. Zinc binds to the N-terminal amino group and His18 imidazole, inducing a kink in the peptide structure, which, in turn, triggers a fibrillization process of the complex, resulting in an amyloid structure most likely responsible for the disruption of the fungal cell.