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.

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.

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.

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.

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.

Two-photon absorption and photoluminescence of colloidal gold nanoparticles and nanoclusters.

This review provides a comprehensive description of nonlinear optical (NLO) properties of gold nanoparticles, which can be used in biological applications. The main focus is placed on two-photon absorption (2PA) and two-photon excited photoluminescence (2PEL) - the processes crucial for multiphoton microscopy, which allows deeper imaging of the material and causes less damage to the biological samples in comparison to conventional (one-photon) microscopy. We present the basics of 2PA measurement techniques and a summary of recent achievements in the understanding of multiphoton excitation and the resulting photoluminescence in gold nanoparticles, both plasmonic ones and small nanoclusters with molecule-like properties. The examples of 2PA applications in bioimaging are also presented, with a comment on future challenges and applications.

Lattice Shrinkage by Incorporation of Recombinant Starmaker-Like Protein within Bioinspired Calcium Carbonate Crystals.

The biological mediation of mineral formation (biomineralization) is realized through diverse organic macromolecules that guide this process in a spatial and temporal manner. Although the role of these molecules in biomineralization is being gradually revealed, the molecular basis of their regulatory function is still poorly understood. In this study, the incorporation and distribution of the model intrinsically disordered starmaker-like (Stm-l) protein, which is active in fish otoliths biomineralization, within calcium carbonate crystals, is revealed. Stm-l promotes crystal nucleation and anisotropic tailoring of crystal morphology. Intracrystalline incorporation of Stm-l protein unexpectedly results in shrinkage (and not expansion, as commonly described in biomineral and bioinspired crystals) of the crystal lattice volume, which is described herein, for the first time, for bioinspired mineralization. A ring pattern was observed in crystals grown for 48 h; this was composed of a protein-enriched region flanked by protein-depleted regions. It can be explained as a result of the Ostwald-like ripening process and intrinsic properties of Stm-l, and bears some analogy to the daily growth layers of the otolith.

Two-photon chiro-optical properties of gold Au25 nanoclusters.

Atomically-precise chiral gold nanoclusters hold promise for an accurate manipulation of chiro-optical properties, both in linear and nonlinear optics regimes. Here, we present the determination of two-photon linear-circular dichroism and two-photon circular dichroism (TPCD) of Au25[(Capt)18]- (where Capt is captopril). TPCD is found to be two orders of magnitude higher than one-photon circular dichroism.

Two-Photon Imaging of 3D Organization of Bimetallic AuAg Nanoclusters in DNA Matrix.

We report on two-photon excitation properties of small silver-doped gold nanoclusters (AuAgNCs) and on their three-dimensional arrangement in a hybrid system composed of DNA liquid crystals (LCs) and AuAgNCs. UV-vis and fluorescence spectroscopy, transmission electron microscopy (TEM), and multiphoton excitation spectroscopy were used to characterize the nanoparticles. We show that AuAgNCs exhibit two-photon excited luminescence (2PL) emission and second-harmonic generation (SHG) and that these properties remain the same in liquid crystalline matrix. The results are described in detail and discussed in the context of possible imaging application of AuAgNC and specific AuAgNCs organization induced by liquid crystalline ordering of DNA molecules.

Linear Optical and Third-Order Nonlinear Optical Properties of Some Fluorenyl- and Triarylamine-Containing Tetracyanobutadiene Derivatives.

The synthesis and characterization of four new tetracyanobutadiene (TCBD) derivatives (1-3 and 2') incorporating 2- or 2,7-fluorenyl and diphenylamino moieties are reported. The electroactivity of 1-3 and 2' was studied by cyclic voltammetry (CV), while the linear optical and (third-order) nonlinear optical (NLO) properties were investigated by electronic spectroscopy and Z-scan studies, respectively. All experimental investigations were rationalized by DFT computations, providing an insight into the electronic structure of these derivatives and on their application potential. We show that these derivatives are nonluminescent in solution at ambient temperatures, but become fluorescent in solvent glasses. This finding constitutes an unprecedented observation for TCBD derivatives. Also, we show by Z-scan studies that these derivatives behave as two-photon absorbers in the near-IR range (800-1050 nm). These third-order NLO properties are discussed and compared with those of their alkynyl precursors (4-6), which have been investigated by two-photon excited fluorescence (TPEF).

Unravelling the Binding Mechanism of a Poly(cationic) Anthracenyl Fluorescent Probe with High Affinity toward Double-Stranded DNA.

We report the synthesis, spectroscopy, and the DNA binding properties of a biocompatible, water-soluble, polycationic two-photon absorbing anthracenyl derivative (Ant-PIm) specifically designed for biorelated applications. Detailed insights into the Ant-PIm-DNA binding interaction are provided by using several spectroscopic approaches, including UV-vis absorption, circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), steady-state, and time-resolved fluorescence techniques. Absorption and fluorescence quantitative data analysis show a strong Ant-PIm-duplex interaction with binding constants of Kf = 4.7 ± 0.2 × 105 M-1, 7.1 ± 0.3 × 105 M-1, and 1.0 ± 0.1 × 106 M-1 at 298, 304, and 310 K, respectively. Spectral changes observed upon DNA binding provide evidence for a complex formation with off-on fluorescence pattern, which can be related to two consecutive binding equilibria. Results of DNA binders displacement and iodide quenching experimental assays unambiguously point to the groove binding mode of Ant-PIm to the DNA-helicate. Thermodynamic and chemical denaturation studies suggest that long-range interactions of hydrophobic nature regulate the association of Ant-PIm with the biopolymer. The ionic strength dependence of the binding constant shows that electrostatic component has an important contribution to the overall Gibbs free energy. FTIR and CD data provide evidence of partial modification of the B-DNA secondary structure, while the increase in the melting temperature clearly indicates the enhancement of the thermal stability of the duplex. Furthermore, the two-photon absorption cross section spectrum determined using the two-photon excited fluorescence (TPEF) technique shows a strong 2PA maximum at 820 nm with a σ2 > 800 GM, which emphasizes the advantageous combination of biological and optical properties possessed by this positively charged bioprobe.

Stabilization of DNA liquid crystals on doping with gold nanorods.

We report on the impact of doping with gold nanorods (NRs) on the formation and stability of DNA liquid crystals (LCs). Cetyl trimethylammonium (CTAB)-stabilized gold NRs were synthesized using the wet chemistry method. Different textures of cholesteric and columnar mesophases, as well as phase transitions, were observed using a polarized light microscope. It was found that liquid crystalline phases formed in the samples were qualitatively the same and the phase appearance sequence was preserved in the samples regardless of the doping. We show that depending on the concentration of gold NRs present in the phase, nanoparticle-doped cholesteric and columnar hexagonal phases existed in wider temperature ranges compared to pure DNA LCs. The potential applications of these liquid crystal-nanoparticle hybrid systems may include the fabrication of new optoelectronic devices and sensors.

Bio-mediated synthesis, characterization and cytotoxicity of gold nanoparticles.

We report here a "green" approach for the synthesis of gold nanoparticles (GNPs) in which the Mentha piperita extract was applied for the bioreduction of chloroauric acid and the stabilization of the formed nanostructures. The obtained GNPs were characterized by UV-Vis absorption spectroscopy and transmission electron microscopy (TEM). The reduction of gold ions with the plant extract leads to the production of nanoparticles with various shapes (spherical, triangular and hexagonal) and sizes (from 10 to 300 nm). The kinetics of the reaction was monitored and various conditions of the synthesis were investigated. As a result, we established protocols optimized towards the synthesis of nanospheres and nanoprisms of gold. The cytotoxic effect of the obtained gold nanoparticles was studied by performing MTT assay, which showed lower cytotoxicity of the biosynthesized GNPs compared to gold nanorods synthesized using the usual seed-mediated growth. The results suggest that the synthesis using plant extracts may be a useful method to produce gold nanostructures for various biological and medical applications.

Interactions of a biocompatible water-soluble anthracenyl polymer derivative with double-stranded DNA.

We have studied the interaction of a polymeric water soluble anthracenyl derivative () with salmon testes DNA. The results from UV-Vis, fluorescence, Fourier transform infrared (FT-IR) and circular dichroism spectroscopies indicate that the groove binding process regulates the interaction between and DNA. The binding constants, calculated by absorption spectroscopy at 298, 304 and 310 K, were equal to 3.2 × 10(5) M(-1), 4.7 × 10(5) M(-1), and 6.6 × 10(5) M(-1) respectively, proving a relatively high affinity of for salmon testes DNA. Results of Hoechst 33258 displacement assays strongly support the groove binding mode of to DNA. The association stoichiometry of the :DNA adduct was found to be 1 for every 5 base pairs. FT-IR spectra, recorded at different /DNA molar ratios, indicate the involvement of the phosphate groups and adenine and thymine DNA bases in the association process. Thermodynamic results suggest that hydrophobic forces regulate the binding of with DNA without excluding some extent of involvement of van der Waals forces and hydrogen bonding arising due to surface binding between the hydrophilic polymeric arms of the ligand and the functional groups positioned on the edge of the groove. The resulting composite biomaterial could constitute a valuable candidate for future biological and/or photonic applications.

Post-synthesis reshaping of gold nanorods using a femtosecond laser.

This work describes the interaction between femtosecond laser pulses (~130 fs, 800 nm) and gold nanorods (NRs) leading to reshaping of the NRs. We focus on the investigation of structural changes of the NRs and the parameters influencing the reshaping, like surface modification using sodium sulphide, laser power and the position of the longitudinal surface plasmon resonance band (l-SPR) with respect to the laser wavelength. A thermogravimetric analysis experiment is performed to examine changes in the composition of NRs upon heating. A new type of banana-shaped NPs is described and the conditions of their appearance are discussed.

A 5-(difluorenyl)-1,10-phenanthroline-based Ru(II) complex as a coating agent for potential multifunctional gold nanoparticles.

The synthesis and photophysical properties of small gold nanoparticles (NPs, AuNP-[Ru-PFF]) surface functionalized by 5-substituted-1,10-phenanthroline-ligand based Ru(II) complexes are described. Luminescence of the grafted and confined Ru(II) complexes is totally quenched on the gold surface. Nonlinear optical properties were determined via Z-scan measurements in the range 600-1300 nm for both the free Ru(II) complex and the related NPs. In the short wavelength range (around 600 nm) the behaviour switches from that of two-photon absorption (2PA) for the complex to saturable absorption for the NPs. 2PA applications such as optical power limiting or two-photon dioxygen sensitization can be anticipated for these nanoplatforms.

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.

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.

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.