Tailoring the interaction between a gold nanocluster and a fluorescent dye by cluster size: creating a toolbox of range-adjustable pH sensors.

We present a novel strategy for tailoring the fluorescent azadioxatriangulenium (KU) dye-based pH sensor to the target pH range by regulating the pK a value of the gold nanoclusters. Based on the correlation between the pK a and surface curvature of ligand-protected nanoparticles, the pK a value of the gold nanoclusters was controlled by size. In particular, three different-sized para-mercaptobenzoic acid (p-MBA) protected gold nanoclusters, Au25(p-MBA)18, Au102(p-MBA)44, and Au210-230(p-MBA)70-80 were used as the regulator for the pH range of the KU response. The negatively charged gold nanoclusters enabled the positively charged KU to bind to the surface, forming a complex and quenching the fluorescence of the KU by the energy transfer process. The fluorescence was restored after adjusting the surface charge of the gold nanocluster by controlling the solution pH. In addition, the KU exhibited a significantly different pH response behaviour for each gold nanocluster. Au210-230(p-MBA)70-80 showed a higher pH response range than Au102(p-MBA)44, which was intuitive. However, Au25(p-MBA)18 showed an unexpectedly high pH response behaviour. pK a titration measurement, molecular dynamics simulations, and essential dynamics analysis showed that small nanoclusters do not follow the scaling between the curvature and the pK a value. Instead, the behaviour is governed by the distribution and interaction of p-MBA ligands on the nanocluster surface. This work presents an effective design strategy for fabricating a range adjustable pH sensor by understanding the protonation behaviour of the ultrasmall gold nanoclusters in an atomic range.

Systematic study of SYBR green chromophore reveals major improvement with one heteroatom difference.

Five nucleic acid binding cyanine dyes were synthesized and their photophysical properties were evaluated. Changing a single heteroatom in the chromophore causes major differences both in brightness and photostability between the dyes. With such alteration, the brightness of the chromophore increased two-fold compared to the one found in SYBR Green I.

Covalent and non-covalent coupling of a Au102 nanocluster with a fluorophore: energy transfer, quenching and intracellular pH sensing.

Interactions between an atomically precise gold nanocluster Au102(p-MBA)44 (p-MBA = para mercaptobenzoic acid) and a fluorescent organic dye molecule (KU, azadioxatriangulenium) are studied. In solution, the constituents form spontaneously a weakly bound complex leading to quenching of fluorescence of the KU dye via energy transfer. The KU can be separated from the complex by lowering pH, leading to recovery of fluorescence, which forms a basis for an optical reversible pH sensor. However, the sensor is not a stable entity, which could be delivered inside cells. For this purpose, a covalently bound hybrid is synthesized by linking the KU dye to the ligand layer of the cluster via an ester bond. Covalent linking facilitates entry of the cluster-dye hybrids into cells via endocytosis. Inside cells, the hybrids accumulate in endosomes where Au102 releases its cargo via hydrolysis of the ester bond. Changes of the local pH inside endosomes regulate reversible fluorescence due to variations in the interactions between the Au102 cluster and the dye. This work presents a concept for delivering reporter molecules into cells by using atomically precise gold nanoclusters as carriers and paves the way for future developments of cluster-reporter sensors for in vivo measurements of e.g. absolute pH values or ion concentrations.

Chemically Selective Imaging of Individual Bonds through Scanning Electron Energy-Loss Spectroscopy: Disulfide Bridges Linking Gold Nanoclusters.

As proof-of-principle of chemically selective, spatially resolved imaging of individual bonds, we carry out electron energy-loss spectroscopy in a scanning transmission electron microscopy instrument on atomically precise, thiolate-coated gold nanoclusters linked with 5,5'-bis(mercaptomethyl)-2,2'-bipyridine dithiol ligands. The images allow the identification of bridging disulfide bonds (R-S-S-R) between clusters, and X-ray photoelectron spectra support the finding.

Dithiol-Induced Oligomerization of Thiol-Protected Gold Nanoclusters.

Controlled synthesis of nanostructure oligomers requires detailed understanding of their wet chemistry and the forces driving the polymerization process. In this paper, we report the main factors affecting the reaction yields of a dithiol-induced synthesis of covalently bound nanocluster dimers and oligomers and present a detailed analysis of possible reaction mechanisms. We synthesize the nanocluster oligomers using monodisperse para-mercaptobenzoic acid (p-MBA)-protected gold nanoclusters with a nominal composition of Au∼250(p-MBA) n to minimize ensemble effects on size, shape, and surface structure. Ligand exchange was performed on the nanoclusters with five different dithiol linkers: 5,5'-bis(mercaptomethyl)-2,2'-bipyridine, 4,4″-thiobisbenzenethiol, benzene-1,4-dithiol, 1,4-benzenedimethanethiol, and dimercaptostilbene. Oligomer yields depend strongly on the used dithiol and on the dithiol-to-nanocluster ratio. Detailed analysis of the reaction yields in combination with simulations suggests that the system reaches a dynamic equilibrium, where ligand exchange happens continuously forming and breaking nanocluster oligomers that are bound together by short chains of disulfide-bridged dithiols. Despite the dynamic nature of the system, dithiol-induced polymerization of nanoclusters is a general and straightforward approach to produce dimers and larger oligomers of thiol-protected nanoclusters. Our work provides physical insight into, offers tools for, and reveals challenges in the controlled synthesis of covalently bound nanoparticle assemblies.

Atomically Precise Nanocluster Assemblies Encapsulating Plasmonic Gold Nanorods.

The self-assembled structures of atomically precise, ligand-protected noble metal nanoclusters leading to encapsulation of plasmonic gold nanorods (GNRs) is presented. Unlike highly sophisticated DNA nanotechnology, this strategically simple hydrogen bonding-directed self-assembly of nanoclusters leads to octahedral nanocrystals encapsulating GNRs. Specifically, the p-mercaptobenzoic acid (pMBA)-protected atomically precise silver nanocluster, Na4 [Ag44 (pMBA)30 ], and pMBA-functionalized GNRs were used. High-resolution transmission and scanning transmission electron tomographic reconstructions suggest that the geometry of the GNR surface is responsible for directing the assembly of silver nanoclusters via H-bonding, leading to octahedral symmetry. The use of water-dispersible gold nanoclusters, Au≈250 (pMBA)n and Au102 (pMBA)44 , also formed layered shells encapsulating GNRs. Such cluster assemblies on colloidal particles are a new category of precision hybrids with diverse possibilities.

Structural characterization of site-modified nanocapsid with monodispersed gold clusters.

Hepatitis E Virus-like particles self-assemble in to noninfectious nanocapsids that are resistant to proteolytic/acidic mucosal delivery conditions. Previously, the nanocapsid was engineered to specifically bind and enter breast cancer cells, where successful tumor targeting was demonstrated in animal models. In the present study, the nanocapsid surface was modified with a solvent-exposed cysteine to conjugate monolayer protected gold nanoclusters (AuNC). Unlike commercially available gold nanoparticles, AuNCs monodisperse in water and are composed of a discrete number of gold atoms, forming a crystalline gold core. Au102 pMBA44 (Au102) was an ideal conjugate given its small 2.5 nm size and detectability in cryoEM. Au102 was bound directly to nanocapsid surface cysteines via direct ligand exchange. In addition, Au102 was functionalized with a maleimide linker (Au102_C6MI) for maleimide-thiol conjugation to nanocapsid cysteines. The AuNC-bound nanocapsid constructs were conjugated in various conditions. We found Au102_C6MI to bind nanocapsid more efficiently, while Au102 remained more soluble over time. Nanocapsids conjugated to Au102_C6MI were imaged in cryoEM for single particle reconstruction to localize AuNC position on the nanocapsid surface. We resolved five unique high intensity volumes that formed a ring-shaped density at the 5-fold symmetry center. This finding was further supported by independent rigid modeling.

Exploring the atomic structure of 1.8nm monolayer-protected gold clusters with aberration-corrected STEM.

Monolayer-protected (MP) Au clusters present attractive quantum systems with a range of potential applications e.g. in catalysis. Knowledge of the atomic structure is needed to obtain a full understanding of their intriguing physical and chemical properties. Here we employed aberration-corrected scanning transmission electron microscopy (ac-STEM), combined with multislice simulations, to make a round-robin investigation of the atomic structure of chemically synthesised clusters with nominal composition Au144(SCH2CH2Ph)60 provided by two different research groups. The MP Au clusters were "weighed" by the atom counting method, based on their integrated intensities in the high angle annular dark field (HAADF) regime and calibrated exponent of the Z dependence. For atomic structure analysis, we compared experimental images of hundreds of clusters, with atomic resolution, against a variety of structural models. Across the size range 123-151 atoms, only 3% of clusters matched the theoretically predicted Au144(SR)60 structure, while a large proportion of the clusters were amorphous (i.e. did not match any model structure). However, a distinct ring-dot feature, characteristic of local icosahedral symmetry, was observed in about 20% of the clusters.

Template-Free Supracolloidal Self-Assembly of Atomically Precise Gold Nanoclusters: From 2D Colloidal Crystals to Spherical Capsids.

We report supracolloidal self-assembly of atomically precise and strictly monodisperse gold nanoclusters involving p-mercaptobenzoic acid ligands (Au102 -pMBA44 ) under aqueous conditions into hexagonally packed monolayer-thick two-dimensional facetted colloidal crystals (thickness 2.7 nm) and their bending to closed shells leading to spherical capsids (d ca. 200 nm), as controlled by solvent conditions. The 2D colloidal assembly is driven in template-free manner by the spontaneous patchiness of the pMBA ligands around the Au102 -pMBA44 nanoclusters preferably towards equatorial plane, thus promoting inter-nanocluster hydrogen bonds and high packing to planar sheets. More generally, the findings encourage to explore atomically precise nanoclusters towards highly controlled colloidal self-assemblies.

Covalently linked multimers of gold nanoclusters Au102(p-MBA)44 and Au∼250(p-MBA)n.

We present the synthesis, separation, and characterization of covalently-bound multimers of para-mercaptobenzoic acid (p-MBA) protected gold nanoclusters. The multimers were synthesized by performing a ligand-exchange reaction of a pre-characterized Au102(p-MBA)44 nanocluster with biphenyl-4,4'-dithiol (BPDT). The reaction products were separated using gel electrophoresis yielding several distinct bands. The bands were analyzed by transmission electron microscopy (TEM) revealing monomer, dimer, and trimer fractions of the nanocluster. TEM analysis of dimers in combination with molecular dynamics simulations suggest that the nanoclusters are covalently bound via a disulfide bridge between BPDT molecules. The linking chemistry is not specific to Au102(p-MBA)44. The same approach yields multimers also for a larger monodisperse p-MBA-protected cluster of approximately 250 gold atoms, Au∼250(p-MBA)n. While the Au102(p-MBA)44 is not plasmonic, the Au∼250(p-MBA)n nanocluster supports localized surface plasmon resonance (LSPR) at 530 nm. Multimers of the Au∼250(p-MBA)n exhibit additional transitions in their UV-vis spectrum at 630 nm and 810 nm, indicating the presence of hybridized LSPR modes. Well-defined structures and relatively small sizes make these systems excellent candidates for connecting ab initio theoretical studies and experimental quantum plasmonics. Moreover, our work opens new possibilities in the controlled synthesis of advanced monodisperse nanocluster superstructures.

Hydrophobic pocket targeting probes for enteroviruses.

Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron microscopes. The probe mildly stabilized the virus particle by increasing the melting temperature by 1-3 degrees, and caused a delay in the uncoating of the virus in the cellular endosomes, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding moiety of the probe was shown to bind to echovirus 1 particle by STD and tr-NOESY NMR methods. Furthermore, binding to echovirus 1 and Coxsackievirus A9, and to a lesser extent to Coxsackie virus B3 was verified by using a gold nanocluster labeled probe by TEM analysis. Molecular modelling suggested that the probe fits the hydrophobic pockets of EV1 and CVA9, but not of CVB3 as expected, correlating well with the variations in the infectivity and stability of the virus particles. EV1 conjugated to the fluorescent dye labeled probe was efficiently internalized into the cells. The virus-fluorescent probe conjugate accumulated in the cytoplasmic endosomes and caused infection starting from 6 hours onwards. Remarkably, before and during the time of replication, the fluorescent probe was seen to leak from the virus-positive endosomes and thus separate from the capsid proteins that were left in the endosomes. These results suggest that, like the physiological hydrophobic content, the probe may be released upon virus uncoating. Our results collectively thus show that the gold and fluorescently labeled probes may be used to track and visualize the studied enteroviruses during the early phases of infection opening new avenues to follow virus uncoating in cells.

Molecule-like photodynamics of Au102(pMBA)44 nanocluster.

Photophysical properties of a water-soluble cluster Au102(pMBA)44 (pMBA = para-mercaptobenzoic acid) are studied by ultrafast time-resolved mid-IR spectroscopy and density functional theory calculations in order to distinguish between molecular and metallic behavior. In the mid-IR transient absorption studies, visible or near-infrared light is used to electronically excite the sample, and the subsequent relaxation is monitored by studying the transient absorption of a vibrational mode in the ligands. Based on these studies, a complete picture of energy relaxation dynamics is obtained: (1) 0.5-1.5 ps electronic relaxation, (2) 6.8 ps vibrational cooling, (3) intersystem crossing from the lowest triplet state to the ground state with a time constant 84 ps, and (4) internal conversion to the ground state with a time constant of ∼3.5 ns. A remarkable finding based on this work is that a large cluster containing 102 metal atoms behaves like a small molecule in a striking contrast to a previously studied slightly larger Au144(SC2H4Ph)60 cluster, which shows relaxation typical for metallic particles. These results therefore establish that the transition between molecular and metallic behavior occurs between Au102 and Au144 species.

Solvation chemistry of water-soluble thiol-protected gold nanocluster Au102 from DOSY NMR spectroscopy and DFT calculations.

The hydrodynamic diameter of Aum(pMBA)n [(m, n) = (102, 44) and (144, 60)] clusters in aqueous media was determined via DOSY NMR spectroscopy. The apparent size of the same (n, m) cluster depends on the counter ion of the deprotonated pMBA(-) ligand as explained by the competing ion-pair strength and hydrogen bonding interactions studied in DFT calculations. The choice of the counter ion affects the surface chemistry and molecular structure at the organic/water interface, which is relevant for biological applications.

4,4-Di-fluoro-2,3;5,6-bis-(tetra-methylene)-4-bora-3a,4a-di-aza-s-indacene (LD540).

The title compound, C18H21BF2N2, is a lipophilic dye based on a BODIPY fluoro-phore backbone, which was developed for microscopic imaging of lipid droplets; the mol-ecule has a planar BODIPY core [dihedral angle between the pyrrole rings = 2.3 (3)°] and two tetra-methyl-ene substituents at the 2,3- and 5,6-positions in a half-chair conformation. One of the tetra-methyl-ene substituents is disordered over two two sets of sites with site occupancies of 0.5. In the crystal, pairs of C-H⋯F inter-actions link the mol-ecules into inversion dimers. Neighbouring dimers are linked by further C-H⋯F inter-actions, forming an infinite array. C-H⋯π and π-π [centroid-centroid distance = 4.360 (3) Å] inter-actions are observed between the BODIPY core and the tetra-methyl-ene substituents of neighbouring dimer pairs.

Site-specific targeting of enterovirus capsid by functionalized monodisperse gold nanoclusters.

Development of precise protocols for accurate site-specific conjugation of monodisperse inorganic nanoparticles to biological material is one of the challenges in contemporary bionanoscience and nanomedicine. We report here a successful site-specific covalent conjugation of functionalized atomically monodisperse gold clusters with 1.5-nm metal cores to viral surfaces. Water-soluble Au102(para-mercaptobenzoic acid)44 clusters, functionalized by maleimide linkers to target cysteines of viral capsid proteins, were synthesized and conjugated to enteroviruses echovirus 1 and coxsackievirus B3. Quantitative analysis of transmission electron microscopy images and the known virus structures showed high affinity and mutual ordering of the bound gold clusters on the viral surface and a clear correlation between the clusters and the targeted cysteine sites close to the viral surface. Infectivity of the viruses was not compromised by loading of several tens of gold clusters per virus. These advances allow for future investigations of the structure-function relations of enteroviruses and enterovirus-related virus-like particles, including their entry mechanisms into cells and uncoating in cellular endosomes.

Vibrational Perturbations and Ligand-Layer Coupling in a Single Crystal of Au144(SC2H4Ph)60 Nanocluster.

We have determined vibrational signatures and optical gap of the Au144(PET)60 (PET: phenylethylthiol, SC2H4Ph) nanocluster solvated in deuterated dichloromethane (DCM-D2, CD2Cl2) and in a single crystal. For crystals, solid-state (13)C NMR and X-ray diffraction were also measured. A revised value of 2200 cm(-1) (0.27 eV) was obtained for the optical gap in both phases. The vibrational spectra of solvated AU144(PET)60 closely resembles that of neat PET, while the crystalline-state spectrum exhibits significant inhomogeneous spectral broadening, frequency shifts, intensity transfer between vibrational modes, and an increase in the overtone and combination transition intensities. Spectral broadening was also observed in the (13)C NMR spectrum. Changes in the intensity are explained due to vibrational coupling of the normal modes induced by the crystal packing, and the vibrational broadening is caused by ligand-environment inhomogeneity in the crystal. This indicates a pseudocrystalline state where the cluster cores are arranged in periodic fashion, while the ligand-layer molecules between the cores form amorphous structures.

Nondestructive size determination of thiol-stabilized gold nanoclusters in solution by diffusion ordered NMR spectroscopy.

Diffusion ordered NMR spectroscopy (DOSY) was used as an analytical tool to estimate the size of thiol-stabilized gold nanoclusters in solution, namely, phenylethanethiol (PET) stabilized Au25(PET)18, Au38(PET)24, and Au144(PET)60. This was achieved by determining the diffusion coefficient and hydrodynamic radius from solution samples that were confirmed to be monodispersed by electrospray ionization mass spectrometry. The average cluster diameters obtained by this technique were estimated to be 1.7, 2.2, and 3.1 nm for the Au25(PET)18, Au38(PET)24, and Au144(PET)60 nanoclusters, respectively, which were shown to agree well with the average diameters of the corresponding single crystal or theoretical structures reported in the literature. Consequently, the DOSY technique is demonstrated to be a potentially valuable nondestructive tool for characterization of nanoparticle mixtures and verifying the purity of product solutions.

An overview of the methods used in the characterisation of natural organic matter (NOM) in relation to drinking water treatment.

Natural organic matter (NOM) is found in all surface, ground and soil waters. During recent decades, reports worldwide show a continuing increase in the color and NOM of the surface water, which has an adverse affect on drinking water purification. For several practical and hygienic reasons, the presence of NOM is undesirable in drinking water. Various technologies have been proposed for NOM removal with varying degrees of success. The properties and amount of NOM, however, can significantly affect the process efficiency. In order to improve and optimise these processes, the characterisation and quantification of NOM at different purification and treatment processes stages is important. It is also important to be able to understand and predict the reactivity of NOM or its fractions in different steps of the treatment. Methods used in the characterisation of NOM include resin adsorption, size exclusion chromatography (SEC), nuclear magnetic resonance (NMR) spectroscopy, and fluorescence spectroscopy. The amount of NOM in water has been predicted with parameters including UV-Vis, total organic carbon (TOC), and specific UV-absorbance (SUVA). Recently, methods by which NOM structures can be more precisely determined have been developed; pyrolysis gas chromatography-mass spectrometry (Py-GC-MS), multidimensional NMR techniques, and Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The present review focuses on the methods used for characterisation and quantification of NOM in relation to drinking water treatment.

(E)-7-(Pyren-1-yl)hept-6-enoic acid.

The title compound, C(23)H(20)O(2), is a precursor of a pyrene-based supra-molecular element for non-covalent attachment to a carbon nanotube. The asymmetric unit contains three independent mol-ecules. The carb-oxy-lic acid group in each of these mol-ecules serves as an inter-molecular hydrogen-bond donor and acceptor, generating the commonly observed double O-H⋯O hydrogen-bond motif in an eight-membered ring. Weaker C-H⋯O, π-π [centroid-centroid distance = 3.968 (4) Å] and C-H⋯π inter-actions are also found in the crystal structure.

N-Benzyl-2,3,4,5,6-penta-fluoro-benz-amide.

In the title compound, C(14)H(8)F(5)NO, the dihedral angle between the planes of the penta-fluoro-phenyl and phenyl rings is 18.34 (5)°. An inter-molecular N-H⋯O hydrogen bond between the amide groups connects these mol-ecules to form an infinite chain through the crystal structure. One weak intermolecular C-H⋯O contact and one π-π interaction [centroid-centroid distance = 3.772 (3) Å] are also involved in crystal structure stabilization between the phenyl rings.