One- and two-photon brightness of proteins interacting with gold. A closer look at gold-insulin conjugates.

Red luminophores displaying large Stokes shift and high-quantum yields are obtained when gold salts are reacted with proteins under strongly alkaline conditions. Although bovine serum albumin (BSA) has mainly been used as a protein template, other attempts to prepare red luminophores have been proposed using other proteins. Here, we report on the structural characterization and nonlinear optical properties of insulin-gold conjugates. Such conjugates display strong luminescence at ∼670 nm with quantum yields that reach 5.4%. They also display long luminescence lifetimes allowing efficient reactive oxygen species generation, with a quantum yield of 1O2 generation reaching 13%. In addition, they exhibit remarkable nonlinear optical properties and in particular a strong two-photon excited fluorescence (TPEF) cross section in the range of 800-1100 nm. By combining experimental studies and time-dependent density functional theory simulations (TD-DFT), we show the formation of insulin-Au(III) conjugates. The interaction of Au(III) ions with the aromatic rings of tyrosine induces charge transfer-like excitation in the visible range. Experimental investigations, together with molecular dynamics simulations of insulin and calculations of electronic properties in a model system, are performed to explore the origin of optical features and the structure-optical property relationship, leading the way to new concepts for nonlinear optics using protein-Au(III) conjugates.

Cytotoxic Staphylococcus aureus PSMα3 inhibits the aggregation of human insulin in vitro.

Phenol-soluble modulins (PSMs) are extracellular short amphipathic peptides secreted by the bacteria Staphylococcus aureus (S. aureus). They play an essential role in the bacterial lifecycle, biofilm formation, and stabilisation. From the PSM family, PSMα3 has been of special interest recently due to its cytotoxicity and highly stable α-helical conformation, which also remains in its amyloid fibrils. In particular, PSMα3 fibrils were shown to be composed of self-associating "sheets" of α-helices oriented perpendicular to the fibril axis, mimicking the architecture of canonical cross-ß fibrils. Therefore, they were called cross-α-fibrils. PSMα3 was synthesised and verified for identity with wild-type sequences (S. aureus). Then, using several experimental techniques, we evaluated its propensity for in vitro aggregation. According to our findings, synthetic PSMα3 (which lacks the N-terminal formyl groups found in bacteria) does not form amyloid fibrils and maintains α-helical conformation in a soluble monomeric form for several days of incubation. We also evaluated the influence of PSMα3 on human insulin fibrillation in vitro, using a variety of experimental approaches in combination with computational molecular studies. First, it was shown that PSMα3 drastically inhibits the fibrillation of human insulin. The anti-fibrillation effect of PSMα3 was concentration-dependent and required a concentration ratio of PSMα3: insulin equal to or above 1 : 100. Molecular modelling revealed that PSMα3 most likely inhibits the production of insulin primary nuclei by competing for residues involved in its dimerization.

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.

Single-Particle Photoluminescence Measures a Heterogeneous Distribution of Differential Circular Absorbance of Gold Nanoparticle Aggregates near Constricted Thioflavin T Molecules.

The chirality of biomacromolecules is critical for their function, but the optical signal of this chirality is small in the visible range. Plasmonic nanoparticles are antennas that can couple to this chiral signal. Here, we examine the molecular-scale mechanism behind the induced circular dichroism of gold nanorods (AuNRs) in solution with insulin fibrils and the fibril-intercalating dye thioflavin T (ThT) with polarization-resolved single-molecule fluorescence and single-particle photoluminescence (PL) imaging. We compared the PL upon excitation by left- and right-handed circularly polarized light to calculate the differential absorbance of AuNRs near insulin fibrils with and without ThT. Overall, our results indicate that AuNRs do not act as chiral absorbers near constricted ThT molecules. Instead, we hypothesize that fibrils promote AuNR aggregation, and this templating is mediated by subtle changes in the solution conditions; under the right conditions, only a few chiral aggregates with significantly higher circular dichroism signal contribute to a large net circular dichroism.

Amyloid engineering - how terminal capping modifies morphology and secondary structure of supramolecular peptide aggregates.

The effects of peptide N- and C-termini on aggregation behavior have been scarcely studied. Herein, we examine (105-115) peptide fragments of transthyretin (TTR) containing various functional groups at both termini and study their impact on the morphology and the secondary structure. We synthesized TTR(105-115) peptides functionalized with α-amino (H-), N-acetyl-α-amino (Ac-) or N,N-dimethyl-α-amino (DiMe-) groups at the N-terminus, and with amide (-NH2) or carboxyl (-OH) functions at the C-terminus. We also investigated quasi-racemic mixtures by mixing the L-enantiomers with the D-enantiomer capped by H- and -NH2 groups. We observed that fibril formation is promoted by the sufficient number of hydrogen bonds at peptides' termini. Moreover, the final morphology of the aggregates can be controlled by the functional groups at the N-terminus. Remarkably, all quasi-racemic mixtures resulted in the robust formation of fibrils. Overall, this work illustrates how modifications of peptide termini may help to engineer supramolecular aggregates with a predicted morphology.

Bioinorganic chemistry of shepherin II complexes helps to fight Candida albicans?

The fungal cell wall and cell membrane are an important target for antifungal therapies, and a needle-like cell wall or membrane disruption may be an entirely novel antifungal mode of action. In this work, we show how the coordination of Zn(II) triggers the antifungal properties of shepherin II, a glycine- and histidine-rich antimicrobial peptide from the root of Capsella bursa-pastoris. We analyze Cu(II) and Zn(II) complexes of this peptide using experimental and theoretical methods, such as: mass spectrometry, potentiometry, UV-Vis and CD spectroscopies, AFM imaging, biological activity tests and DFT calculations in order to understand the correlation between their metal binding mode, structure, morphology and biological activity. We observe that Zn(II) coordinates to Shep II and causes a structural change, resulting in fibril formation, what has a pronounced biological consequence - a strong anticandidal activity. This phenomenon was observed neither for the peptide itself, nor for its copper(II) complex. The Zn(II) - shepherin II complex can be considered as a starting point for further anticandidal drug discovery, which is extremely important in the era of increasing antifungal drug resistance.

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.

Chitosan oligosaccharides inhibit the fibrillation of insulin and disassemble its preformed fibrils.

In the current study, we first established that chitosan oligosaccharides (COS) have significant anti-fibrillogenic and fibril-destabilising effects on bovine insulin in vitro that proportionally expand with concentration growth. The obtained data were supported by the Thioflavin T (ThT) assay, circular dichroism (CD), attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy, and atomic force microscopy (AFM). Interestingly, coincubation of insulin with COS in the ratio of 1 to 10 over 48 h at 37 °C leads to full prevention of insulin aggregation, and in the case of preformed fibrils, results in their destabilisation and disaggregation. Moreover, both a cationic polymer of allylamine (PAH) and a sulphated oligosaccharide (CROS) prepared from carrageenan had no inhibitory effect on insulin amyloid formation. Thus, we proposed that COS modulates insulin amyloid formation due to the presence of linear sugar units, the degree of polymerization, and the free amino group providing a positive charge. These findings highlight the potential implications of COS as a promising substance for the treatment of insulin-dependent diabetes mellitus and localised insulin-derived amyloidosis and, moreover, provide a new insight into the mechanism of the anti-diabetic and antitoxic properties of chitosan and chitosan-based agents.

Single Gold Nanobipyramids Sensing the Chirality of Amyloids.

Plasmonic nanoparticles, due to their sensitivity to small changes in their closest environment and plasmon resonance, can sense the chirality of the surrounding molecules. Therefore, plasmonic nanoparticles can be applied as a next-generation biosensor for peptides or proteins. In this work, we explore the interaction between chiral, ordered protein aggregates (amyloids) and small gold nanobipyramids. We show how the morphology, structure, and chiroptical properties of amyloids induce circular dichroism in the plasmon resonance wavelengths from individual plasmonic nanoparticles upon binding to the chiral amyloid template. Moreover, using the data from microscopic and spectroscopic analyses of formed heterostructures, we propose the most probable mechanism behind the induction of chirality in this system and discuss which specific feature of insulin protein aggregates is sensed by nanobipyramids.

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) Induces Fibril Formation and Antifungal Activity in Shepherin I, An Antimicrobial Peptide from Capsella bursa-pastoris.

Shepherin I is a glycine- and histidine-rich antimicrobial peptide from the root of a shepherd's purse, whose antimicrobial activity was suggested to be enhanced by the presence of Zn(II) ions. We describe Zn(II) and Cu(II) complexes of this peptide, aiming to understand the correlation between their metal binding mode, structure, morphology, and biological activity. We observe a logical sequence of phenomena, each of which is the result of the previous one: (i) Zn(II) coordinates to shepherin I, (ii) causes a structural change, which, in turn, (iii) results in fibril formation. Eventually, this chain of structural changes has a (iv) biological consequence: The shepherin I-Zn(II) fibrils are highly antifungal. What is of particular interest, both fibril formation and strong anticandidal activity are only observed for the shepherin I-Zn(II) complex, linking its structural rearrangement that occurs after metal binding with its morphology and biological activity.

Solvent-Induced Aggregation of Self-Assembled Copper-Cysteine Nanoparticles Reacted with Glutathione: Enhancing Linear and Nonlinear Optical Properties.

Copper-thiolate self-assembly nanostructures are a unique class of nanomaterials because of their interesting properties such as hierarchical structures, luminescence, and large nonlinear optical efficiency. Herein, we synthesized biomolecule cysteine (Cys) and glutathione (GSH) capped sub-100 nm self-assembly nanoparticles (Cu-Cys-GSH NPs) with red fluorescence. The as-synthesized NPs show high emission enhancement in the presence of ethanol, caused by the aggregation-induced emission. We correlated the structure and optical properties of Cu-Cys-GSH NPs by measuring the mass, morphology, and surface charge as well as their two-photon excited fluorescence cross-section (σ2PEPL), two-photon absorption cross-section (σTPA) and first hyperpolarizability (ß) of Cu-Cys-GSH NPs in water and water-ethanol using near-infrared wavelength. We found a high ß value as (77 ± 10) × 10-28 esu (in water) compared to the reference medium water. The estimated values of σ2PEPL and σTPA are found to be (13 ± 2) GM and (1.4 ± 0.2) × 104 GM, respectively. We hope our investigations of linear and nonlinear optical properties of copper-thiolate self-assemblies in water and its solvent-induced aggregates will open up new possibilities in designing self-assembled systems for many applications including sensing, drug delivery, and catalysis.

Polarization-sensitive Two-photon Microscopy for a Label-free Amyloid Structural Characterization.

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue penetration, efficient operation in densely packed systems, and reduced angular photoselection of fluorophores. Thus, the introduction of polarization analysis in two-photon fluorescence microscopy (2PFM) provides a more precise determination of molecular organization in a sample compared to standard imaging methods based on linear optical processes. In this work, we focus on polarization-sensitive 2PFM (ps-2PFM) and its application in the determination of molecular ordering within complex bio-structures-amyloid spherulites. Neurodegenerative diseases such as Alzheimer's or Parkinson's are often diagnosed through the detection of amyloids-protein aggregates formed due to an impaired protein misfolding process. Exploring their structure leads to a better understanding of their creation pathway and consequently, to developing more sensitive diagnostic methods. This paper presents the ps-2PFM adapted for the determination of local fibril ordering inside the bovine insulin spherulites and spherical amyloidogenic protein aggregates. Moreover, we prove that the proposed technique can resolve the three-dimensional organization of fibrils inside the spherulite.

SARS-CoV-2 Virus-like Particles with Plasmonic Au Cores and S1-Spike Protein Coronas.

The COVID-19 pandemic has stimulated the scientific world to intensify virus-related studies aimed at the development of quick and safe ways of detecting viruses in the human body, studying the virus-antibody and virus-cell interactions, and designing nanocarriers for targeted antiviral therapies. However, research on dangerous viruses can only be performed in certified laboratories that follow strict safety procedures. Thus, developing deactivated virus constructs or safe-to-use virus-like objects, which imitate real viruses and allow performing virus-related studies in any research laboratory, constitutes an important scientific challenge. Such species, called virus-like particles (VLPs), contain instead of capsids with viral DNA/RNA empty or synthetic cores with real virus proteins attached to them. We have developed a method for the preparation of VLPs imitating the virus responsible for the COVID-19 disease: the SARS-CoV-2. The particles have Au cores surrounded by "coronas" of S1 domains of the virus's spike protein. Importantly, they are safe to use and specifically interact with SARS-CoV-2 antibodies. Moreover, Au cores exhibit localized surface plasmon resonance (LSPR), which makes the synthesized VLPs suitable for biosensing applications. During the studies, the effect allowed us to visualize the interaction between the VLPs and the antibodies and identify the characteristic vibrational signals. What is more, additional functionalization of the particles with a fluorescent label revealed their potential in studying specific virus-related interactions. Notably, the universal character of the developed synthesis method makes it potentially applicable for fabricating VLPs imitating other life-threatening viruses.

Strong fluorescence-detected two-photon circular dichroism of chiral gold nanoclusters.

Progress in syntheses and understanding of the intriguing properties of chiral noble metal nanoclusters sparks interest to extend investigations of their chiroptical response to the nonlinear optics regime. We present a quantitative determination of two-photon circular dichroism of chiral gold nanoclusters with ATT and L- or D-Arg ligands (ATT = 6-aza-2-thiotymine and Arg = arginine). Introduction of arginine ligands enables the formation of two enantiomers of the nanoclusters, with strong chiroptical effects in both linear and nonlinear regime. We present two-photon absorption and luminescent properties measured in a wide range of wavelengths, with the two-photon absorption cross section reaching 1743 GM and two-photon brightness ∼1102 GM at 825 nm. We report strong, 245-fold enhancement of the two-photon circular dichroism of nanoclusters with respect to the one-photon absorption counterpart - the dissymmetry factor. The presence of multiple advantages of nanoclusters: high fluorescence quantum yield, strong nonlinear optical properties and well-controlled chirality is a powerful combination for applications of such clusters in multiphoton microscopy.

Crown Ether-Capped Gold Nanoclusters as a Multimodal Platform for Bioimaging.

The distinct polarity of biomolecule surfaces plays a pivotal role in their biochemistry and functions as it is involved in numerous processes, such as folding, aggregation, or denaturation. Therefore, there is a need to image both hydrophilic and hydrophobic bio-interfaces with markers of distinct responses to hydrophobic and hydrophilic environments. In this work, we present a synthesis, characterization, and application of ultrasmall gold nanoclusters capped with a 12-crown-4 ligand. The nanoclusters present an amphiphilic character and can be successfully transferred between aqueous and organic solvents and have their physicochemical integrity retained. They can serve as probes for multimodal bioimaging with light (as they emit near-infrared luminescence) and electron microscopy (due to the high electron density of gold). In this work, we used protein superstructures, namely, amyloid spherulites, as a hydrophobic surface model and individual amyloid fibrils with a mixed hydrophobicity profile. Our nanoclusters spontaneously stained densely packed amyloid spherulites as observed under fluorescence microscopy, which is limited for hydrophilic markers. Moreover, our clusters revealed structural features of individual amyloid fibrils at a nanoscale as observed under a transmission electron microscope. We show the potential of crown ether-capped gold nanoclusters in multimodal structural characterization of bio-interfaces where the amphiphilic character of the supramolecular ligand is required.

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.

Gold-Doping Effect on Two-Photon Absorption and Luminescence of Atomically Precise Silver Ligated Nanoclusters.

Noble metal nanoclusters allow for the atomically-precise control of their composition. However, to create nanoclusters with pre-defined optical properties, comprehensive description of their structure-property relation is required. Here, we report the gold atom doping impact on one-photon and two-photon absorption (TPA) and luminescence properties of ligated silver nanoclusters via combined experimental studies and time-dependent density functional theory simulations (TD-DFT). We synthesized a series of Ag25-x Aux (DMBT)18 nanoclusters where x=0, 1 and 5-10. For Ag24 Au1 (DMBT)18 we demonstrate that the presence of the central Au dopant strongly influences linear and non-linear optical properties, increasing photoluminescence quantum yield and two-photon brightness, with respect to undoped silver nanoclusters. With improved TPA and luminescence, atomically-precise AuAg alloys presented in our work can serve as robust luminescent probes e.g. for bioimaging in the second biological window.

One- and Two-Photon Excited Autofluorescence of Lysozyme Amyloids.

Autofluorescence properties of amyloid fibrils are of much interest but, to date, the attention has been given mostly to one-photon excited fluorescence (1PEF), while the two-photon excited fluorescence (2PEF) properties of amyloids are much less explored. We investigate 1PEF and 2PEF of hen egg-white lysozyme (HEWL) in the form of monomers and fibrils. HEWL monomers feature some autofluorescence, which is enhanced in the case of fibrils. Moreover, by varying NaCl content, we introduce changes to fibrils morphology and show how the increase of the salt concentration is linked with an increase of 1PEF and 2PEF intensities. Interestingly, we observe 2PEF emission red-shifted in comparison to 1PEF. We confirm the presence of different relaxation pathways upon one- or two-photon excitation by different lifetimes of the fluorescence decays. Finally, we correlate the changes in optical properties of HEWL fibrils and monomers with salt-mediated changes in their morphology and the secondary structure.

Plasmonic Enhancement of Two-Photon Excited Luminescence of Gold Nanoclusters.

Plasmonic-enhanced luminescence of single molecules enables imaging and detection of low quantities of fluorophores, down to individual molecules. In this work, we present two-photon excited luminescence of single gold nanoclusters, Au18(SG)14, in close proximity to bare gold nanorods (AuNRs). We observed 25-times enhanced emission of gold nanoclusters (AuNCs) in near infrared region, which was mainly attributed to the resonant excitation of localized surface plasmon resonance (LSPR) of AuNRs and spectral overlap of LSPR band with photoluminescence of AuNCs. This work is an initial step in application of combined nanoparticles: gold nanorods and ultrasmall nanoclusters in a wide range of multiphoton imaging and biosensing applications.