The rising number of antibiotic-resistant bacteria requires alternative antibacterial substances as therapeutics. However, besides a strong effect against bacteria, they should not exhibit cytotoxicity towards human cells. Typically, both properties are tested in separate in vitro experiments that do not take into account naturally occurring interactions. To analyze whether the bacteria-cell interaction influences the results of antibacterial and cytotoxic testing, surfactant-free antibacterial silver-gold alloy nanoparticles produced by laser ablation in liquid were examined both separately and in a straightforward coculture setup. Whereas different commensal bacteria and human skin cells exhibited reduced metabolic activity at comparable concentrations when treated separately, their response completely differed when analyzed in a combined coculture. For the combination of oral keratinocytes and Staphylococcus aureus, reduced cytotoxicity with increasing cell numbers, but a similar antibacterial effect quantified by standard plate counting could be observed. In contrast, coculture of keratinocytes and Porphyromonas gingivalis resulted in a complete absence of cytotoxicity and antibacterial effect, probably due to bacterial invasion of cells. These results clearly showed that the bacteria-cell interaction greatly influences the results of antibacterial and cytotoxic testing and highlight the importance of more complex in vitro experiments to reliably characterize novel antibacterial substances for efficient clinical translation. Typically, antibacterial and cytotoxic properties of novel antibacterial substances are tested in separate experiments that do not consider naturally interactions between bacteria and cells. However, the results of this study showed that the bacteria-cell interaction greatly influences both properties and should be taken into account for in vitro testing.+image

Staphylococcus aureus biofilm-associated infections are a common complication in modern medicine. Due to inherent resilience of biofilms to antibiotics and the rising number of antibiotic-resistant bacterial strains, new treatment options are required. For this purpose, ultrapure, spherical silver–gold-alloy nanoparticles with homogenous elemental distribution were synthesized by laser ablation in liquids and analyzed for their antibacterial activity on different stages of S. aureus biofilm formation as well as for different viability parameters. First, the effect of nanoparticles against planktonic bacteria was tested with metabolic activity measurements. Next, nanoparticles were incubated with differently matured S. aureus biofilms, which were then analyzed by metabolic activity measurements and three dimensional live/dead fluorescent staining to determine biofilm volume and membrane integrity. It could be shown that AgAu NPs exhibit antibacterial properties against planktonic bacteria but also against early-stage and even mature biofilms, with a complete diffusion through the biofilm matrix. Furthermore, AgAu NPs primarily targeted metabolic activity, to a smaller extend membrane integrity, but not the biofilm volume. Additional molecular analyses using qRT-PCR confirmed the influence on different metabolic pathways, like glycolysis, stress response and biofilm formation. As this shows clear similarities to the mechanism of pure silver ions, the results strengthen silver ions to be the major antibacterial agent of the synthesized nanoparticles. In summary, the results of this study provide initial evidence of promising anti-biofilm characteristics of silver–gold-alloy nanoparticles and support the importance of further translation-oriented analyses in the future.

The bactericidal effects of silver nanoparticles (Ag NPs) against infectious strains of multiresistant bacteria is a well-studied phenomenon, highly relevant for many researchers and clinicians battling bacterial infections. However, little is known about the uptake of the Ag NPs into the bacteria, the related uptake mechanisms, and how they are connected to antimicrobial activity. Even less information is available on AgAu alloy NPs uptake. In this work, the interactions between colloidal silver-gold alloy nanoparticles (AgAu NPs) and Staphylococcus aureus (S. aureus) using advanced electron microscopy methods are studied. The localization of the nanoparticles is monitored on the membrane and inside the bacterial cells and the elemental compositions of intra- and extracellular nanoparticle species. The findings reveal the formation of pure silver nanoparticles with diameters smaller than 10 nm inside the bacteria, even though those particles are not present in the original colloid. This finding is explained by a local RElease PEnetration Reduction (REPER) mechanism of silver cations emitted from the AgAu nanoparticles, emphasized by the localization of the AgAu nanoparticles on the bacterial membrane by aptamer targeting ligands. These findings can deepen the understanding of the antimicrobial effect of nanosilver, where the microbes are defusing the attacking silver ions via their reduction, and aid in the development of suitable therapeutic approaches.

Catalytic activity and toxicity of mixed-metal nanoparticles have been shown to correlate and are known to be dependent on surface composition. The surface chemistry of the fully inorganic, ligand-free silver-gold alloy nanoparticle molar fraction series, is highly interesting for applications in heterogeneous catalysis, which is determined by active surface sites which are also relevant for understanding their dissolution behavior in biomedically-relevant ion-release scenarios. However, such information has never been systematically obtained for colloidal nanoparticles without organic surface ligands and has to date, not been analyzed in a surface-normalized manner to exclude density effects. For this, we used detailed electrochemical measurements based on cyclic voltammetry to systematically analyze the redox chemistry of particle-surface-normalized gold-silver alloy nanoparticles with varying gold molar fractions. The study addressed a broad range of gold molar fractions (Ag90Au10, Ag80Au20, Ag70Au30, Ag50Au50, Ag40Au60, and Ag20Au80) as well as monometallic Ag and Au nanoparticle controls. Oxygen reduction reaction (ORR) measurements in O-2 saturated 0.1 M KOH revealed a linear reduction of the overpotential with increasing gold content on the surface, probably attributed to the higher ORR activity of gold over silver, verified by monometallic Ag and Au controls. These findings were complemented by detailed XPS studies revealing an accumulation of the minor constituent of the alloy on the surface, e.g., silver surface enrichment in gold-rich particles. Furthermore, highly oxidized Ag surface site enrichment was detected after the ORR reaction, most pronounced in gold-rich alloys. Further, detailed CV studies at acidic pH, analyzing the position, onset potential, and peak integrals of silver oxidation and silver reduction peaks revealed particularly low reactivity and high chemical stability of the equimolar Au50Ag50 composition, a phenomenon attributed to the outstanding thermodynamic, entropically driven, stabilization arising at this composition.

Biosensors are key components for the early detection of diseases. As such, a higher sensitivity allows for an earlier detection or even detection before symptoms form, as most pathogens and biomarkers have extremely small concentrations. Here we present an ultra-sensitive biosensor based on the scattering of terahertz waves in a spoof surface plasmon polariton resonator on bacteria. While the strongly bound wave and the high-quality factor of the resonator allow a sensitivity in the order of single bacteria, a high selectivity – the other key feature of a good biosensor – is provided by a surface functionalization with aptamers.

Gold-silver alloy nanoparticles are interesting for multiple applications, including heterogeneous catalysis, optical sensing, and antimicrobial properties. The inert element gold acts as a stabilizer for silver to prevent particle corrosion, or conversely, to control the release kinetics of antimicrobial silver ions for long-term efficiency at minimum cytotoxicity. However, little is known about the kinetics of silver ion leaching from bimetallic nanoparticles and how it is correlated with silver content, especially not on a single-particle level. To characterize the kinetics of silver ion release from gold-silver alloy nanoparticles, we employed a combination of electron microscopy and single-particle hyperspectral imaging with an acquisition speed fast enough to capture the irreversible silver ion leaching. Single-particle leaching profiles revealed a reduction in silver ion leaching rate due to the alloying with gold as well as two leaching stages, with a large heterogeneity in rate constants. We modeled the initial leaching stage as a shrinking-particle with a rate constant that exponentially depends on the silver content. The second, slower leaching stage is controlled by the electrochemical oxidation potential of the alloy being steadily increased by the change in relative gold content and diffusion of silver atoms through the lattice. Interestingly, individual nanoparticles with similar sizes and compositions exhibited completely different silver ion leaching yields. Most nanoparticles released silver completely, but 25% of them appeared to arrest leaching. Additionally, nanoparticles became slightly porous. Alloy nanoparticles, produced by scalable laser ablation in liquid, together with kinetic studies of silver ion leaching, provide an approach to design the durability or bioactivity of alloy nanoparticles.

Nano-bio-conjugates, featuring noble metal gold-silver alloy nanoparticles, represent a versatile tool in diagnostics and therapeutics due to their plasmonic and antimicrobial properties tunable by the particle's gold molar fraction. However, little is known about how the binding of thiolated biomolecules to noble metal nanoparticles is influenced by the fraction of gold and silver atoms on the nanoparticle's surface and to which extend this would affect the functionality of the conjugated biomolecules. In this work, we generated gold-silver alloy nanoparticles with average diameters of 7-8 nm using the modern, surfactant-free laser ablation in liquids (LAL) synthesis approach. We conjugated them with thiolated miniStrep aptamer ligands at well-controlled aptamer-to-nanoparticle surface area ratios with maxima between 12 and 27 pmol aptamer/cm2 particle surface area. The results revealed a clear correlation between surface coverage and the nanoparticles' nominal gold/silver ratio, with maximum coverage reached for gold-rich alloys and a pronounced maximum for silver-rich alloys. However, the conjugates' functionality, evaluated by binding of streptavidin, was surprisingly robust and hardly affected by the nominal composition. However, 1.5 times higher surface coverage was needed to obtain maximum functionality in the silver-rich conjugates. Based on these results, it may be concluded that the nominal composition of gold-silver alloy nano-bioconjugates is freely tunable without a pronounced impact on the attached ligands' functionality, a finding highly relevant for the flexible design of nano-bio-conjugates for future biomedical applications. This study's results may facilitate the design of alloy nano-bio-conjugates for future applications in therapeutics and diagnostics.

Bimetallic silver-gold alloy nanoparticles on zirconia with varying Ag/Au ratios were designed by a rational approach and tested as catalysts for the selective oxidation of the promising biomass platform molecule 5-(hydroxymethyl)furfural (HMF). For this purpose, colloidal AgxAu10-x particles with molar compositions x=1/3/5/7/9 were prepared by laser ablation in liquids, a surfactant-free method for the preparation of highly pure nanoparticles, before adsorption on zirconia. In-depth characterization of the supported catalysts evidenced alloyed nanoparticles with distinct trends of the surface and bulk composition depending on the overall Ag/Au molar ratio as determined by X-ray photoelectron spectroscopy (XPS) and X-ray absorption spectroscopy (XAS), respectively. To uncover the synergistic effect of the Ag/Au ratio, the catalysts were further studied in terms of the catalytic activity and selectivity in HMF oxidation. Either the aldehyde moiety or both functional groups of HMF were selectively oxidized depending on the Ag/Au composition resulting in 5-hydroxymethyl-2-furan-carboxylic acid (HFCA) or 2,5-furandicarboxylic acid (FDCA), respectively. Optimization of the reaction conditions allowed the quantitative production of HFCA over most catalysts, also after re-use. Only gold rich catalysts Ag1Au9/ZrO2 and particularly Ag3Au7/ZrO2 were highly active in FDCA synthesis. While Ag3Au7/ZrO2 deactivated upon re-use due to sintering, no structural changes were observed for the other catalysts and all catalysts were stable against metal leaching. The present work thus provides fundamental insights into the synergistic effect of Ag and Au in alloyed nanoparticles as active and stable catalysts for the oxidation of HMF.

Modification of the size and phase composition of magnetic oxide nanomaterials dispersed in liquids by laser synthesis and processing of colloids has high implications for applications in biomedicine, catalysis and for nanoparticle-polymer composites. Controlling these properties for ternary oxides, however, is challenging with typical additives like salts and ligands and can lead to unwanted byproducts and various phases. In our study, we demonstrate how additive-free pulsed laser post-processing (LPP) of colloidal yttrium iron oxide nanoparticles using high repetition rates and power at 355 nm laser wavelength can be used for phase transformation and phase purification of the garnet structure by variation of the laser fluence as well as the applied energy dose. Furthermore, LPP allows particle size modification between 5 nm (ps laser) and 20 nm (ns laser) and significant increase of the monodispersity. Resulting colloidal nanoparticles are investigated regarding their size, structure and temperature-dependent magnetic properties.