Negative Charge-Carrying Glycans Attached to Exosomes as Novel Liquid Biopsy Marker.

Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more nega tive zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.

Correlative atomic force microscopy and scanning electron microscopy of bacteria-diamond-metal nanocomposites.

Research investigating the interface between biological organisms and nanomaterials nowadays requires multi-faceted microscopic methods to elucidate the interaction mechanisms and effects. Here we describe a novel approach and methodology correlating data from an atomic force microscope inside a scanning electron microscope (AFM-in-SEM). This approach is demonstrated on bacteria-diamond-metal nanocomposite samples relevant in current life science research. We describe a procedure for preparing such multi-component test samples containing E. coli bacteria and chitosan-coated hydrogenated nanodiamonds decorated with silver nanoparticles on a carbon-coated gold grid. Microscopic topography information (AFM) is combined with chemical, material, and morphological information (SEM using SE and BSE at varied acceleration voltages) from the same region of interest and processed to create 3D correlative probe-electron microscopy (CPEM) images. We also establish a novel 3D RGB color image algorithm for merging multiple SE/BSE data from SEM with the AFM surface topography data which provides additional information about microscopic interaction of the diamond-metal nanocomposite with bacteria, not achievable by individual analyses. The methodology of CPEM data interpretation is independently corroborated by further in-situ (EDS) and ex-situ (micro-Raman) chemical characterization as well as by force volume AFM analysis. We also discuss the broader applicability and benefits of the methodology for life science research.

Absolute energy levels in nanodiamonds of different origins and surface chemistries.

Nanodiamonds (NDs) are versatile, broadly available nanomaterials with a set of features highly attractive for applications from biology over energy harvesting to quantum technologies. Via synthesis and surface chemistry, NDs can be tuned from the sub-micron to the single-digit size, from conductive to insulating, from hydrophobic to hydrophilic, and from positively to negatively charged surface by simple annealing processes. Such ND diversity makes it difficult to understand and take advantage of their electronic properties. Here we present a systematic correlated study of structural and electronic properties of NDs with different origins and surface terminations. The absolute energy level diagrams are obtained by the combination of optical (UV-vis) and photoelectron (UPS) spectroscopies, Kelvin probe measurements, and energy-resolved electrochemical impedance spectroscopy (ER-EIS). The energy levels and density of states in the bandgap of NDs are correlated with the surface chemistry and structure characterized by FTIR and Raman spectroscopy. We show profound differences in energy band shifts (by up to 3 eV), Fermi level position (from p-type to n-type), electron affinity (from +0.5 eV to -2.2 eV), optical band gap (5.2 eV to 5.5 eV), band gap states (tail or mid-gap), and electrical conductivity depending on the high-pressure, high-temperature and detonation origin of NDs as well as on the effects of NDs' oxidation, hydrogenation, sp2/sp3 carbon phases and surface adsorbates. These data are fundamental for understanding and designing NDs' optoelectrochemical functional mechanisms in diverse application areas.

Non-contact non-resonant atomic force microscopy method for measurements of highly mobile molecules and nanoparticles.

Atomic force microscopy (AFM) is nowadays indispensable versatile scanning probe method widely employed for fundamental and applied research in physics, chemistry, biology as well as industrial metrology. Conventional AFM systems can operate in various environments such as ultra-high vacuum, electrolyte solutions, or controlled gas atmosphere. Measurements in ambient air are prevalent due to their technical simplicity; however, there are drawbacks such as formation of water meniscus that greatly increases attractive interaction (adhesion) between the tip and the sample, reduced spatial resolution, and too strong interactions leading to tip and/or sample modifications. Here we show how the attractive forces in AFM under ambient conditions can be used with advantage to probe surface properties in a very sensitive way even on highly mobile molecules and nanoparticles. We introduce a stable non-contact non-resonant (NCNR) AFM method which enables to reliably perform measurements in the attractive force regime even in air by controlling the tip position in the intimate surface vicinity without touching it. We demonstrate proof-of-concept results on helicene-based macrocycles, DNA on mica, and nanodiamonds on SiO2. We compare the results with other conventional AFM regimes, showing NCNR advantages such as higher spatial resolution, reduced tip contamination, and negligible sample modification. We analyze principle physical and chemical mechanisms influencing the measurements, discuss issues of stability and various possible method implementations. We explain how the NCNR method can be applied in any AFM system by a mere software modification. The method thus opens a new research field for measurements of highly sensitive and mobile nanoscale objects under air and other environments.

Creation and Plasmon-Assisted Photosensitization of Annealed Z-Schemes for Sunlight-Only Water Splitting.

Solely light-induced water splitting represents a promising avenue for a carbon-free energy future, based on reliable energy sources. Such processes can be performed using coupled semiconductor materials (the so-called direct Z-scheme design) that facilitate spatial separation of (photo)excited electrons and holes, prevent their recombination, and allow water-splitting half-reactions proceeding at each corresponding semiconductor side. In this work, we proposed and prepared a specific structure, based on WO3g-x/CdWO4/CdS coupled semiconductors, created by annealing of a common WO3/CdS direct Z-scheme. WO3-x/CdWO4/CdS flakes were further combined with a plasmon-active grating for the creation of the so-called artificial leaf design, making possible complete utilization of the sunlight spectrum. The proposed structure enables water splitting with high production of stoichiometric amounts of oxygen and hydrogen without undesirable catalyst photodegradation. Several control experiments confirm the creation of electrons and holes participating in the water splitting half-reaction in a spatially selective manner.

Energy transfer between Si nanocrystals and protoporphyrin molecules as a function of distance, orientation and size.

The interaction between protoporphyrin molecules (donor) and Si nanocrystals (acceptor) up to 2.5 nm for varying distances and orientations is studied by DFT, semi-empirical and TDDFT methods. Simulations show an effect on electronic structure, indicative of electron charge transfer in parallel orientation and small distances and nonelectron energy transfer for different orientations and larger distances. An absorption-emission spectral overlap is observed. The calculations of coupling and energy transfer rates show a distance dependence typical of fluorescence resonance energy transfer (FRET) in the long range, while in the short and ultra-short range the distance dependence indicates electron transfer in addition to FRET. The Si NCs with the smallest size yield larger couplings than the larger nanocrystals. The PPIX/Si NC coupling was enhanced by adding a plasmon nanoparticle as a bridge in the donor-acceptor system. Results using Au nanoparticles show increased energy transfer rates up to 104 and lower distance dependence.

Polarization Controlled Assembly of Ultrathin Thiorphan Nanostructures on ZnO Surface Facets.

Despite the importance of thiorphan as a small molecule with vital biological roles, its interactions with zinc oxide (ZnO) nanomaterials that are prospective in drug delivery and theranostic applications have not yet been sufficiently explored. Here the impact of surface polarity of different ZnO facets on thiorphan adsorption is studied both experimentally by atomic force microscopy (AFM) and angle resolved X-ray photoelectron spectroscopy (XPS) and theoretically by force field molecular dynamics (FFMD) and density functional tight binding simulations (DFTB). Polar ZnO surfaces cause the formation of thiorphan nanodots, where the size of the nanodots depends on the direction of dipoles: small (4 nm) nanodots are formed on Zn-face ZnO, while large (25 nm) nanodots are formed on O-face ZnO. Nonpolar ZnO surfaces cause self-assembly into layered nanoislands with characteristic 4 nm layer thickness, which subsequently merge into rigid nanolayers. The self-assembly is shown to be controlled solely by the effect of surface dipole electric field orientation and magnitude, whereas effects of surface chemistry or solution are negligible. The results thus also show a way for controlling the assembly of thiorphan and other molecular nanomaterials for diverse applications.

Helicene-based π-conjugated macrocycles: their synthesis, properties, chirality and self-assembly into molecular stripes on a graphite surface.

Fully aromatic helicenes are attractive building blocks for the construction of inherently chiral π-conjugated macrocyclic nanocarbons. These hitherto rare molecular architectures are envisaged to exhibit remarkable (chir)optical properties, self-assembly, charge/spin transport, induced ring current or a fascinating Möbius topology. Here the synthesis of helically chiral macrocycles that combine angular dibenzo[5]helicene units as corners and linear trans-stilbene-4,4'-diyl linkers as edges is reported. By subjecting a racemic or enantiopure divinyl derivative of dibenzo[5]helicene to olefin metathesis, which was catalysed by a 2nd generation Piers catalyst under kinetic control, a π-conjugated helicene cyclic trimer (33%) and a tetramer (22%) were obtained, which were separated by GPC. Combining racemic/asymmetric synthesis with the resolution of enantiomers/diastereomers by SFC/HPLC on a chiral column, both homochiral (+)-(M,M,M)/(-)-(P,P,P) and heterochiral (+)-(M,M,P)/(-)-(M,P,P) stereoisomers of the helicene cyclic trimer could be obtained in an enantio- and diastereomerically enriched form. The complete energy profile of their interconversion was compiled on the basis of kinetic measurements and numerical solution of the proposed kinetic model. In equilibrium, the heterochiral diastereomer predominates over the homochiral one (ca. 75 : 25 at 76 °C). π-Conjugation along a large, twisted circuit in the helicene cyclic trimer is rather disrupted, stabilising this formally antiaromatic molecule. Using an optimised PeakForce mode of ambient AFM, the self-assembly of otherwise highly mobile stereoisomers of the helicene cyclic trimer on the HOPG surface could be studied. Irrespective of the stereochemistry, strong preferences for the edge-to-edge interaction of these macrocycles were found to form very long parallel 1D molecular stripes in ordered 2D nanocrystals, a result also supported by molecular dynamics simulations. Six trityl groups, initially introduced to the macrocycle to enhance solubility, serve as a key "molecular Velcro" system in the self-assembly of macrocycles to maximise their mutual van der Waals interactions.

Coating Ti6Al4V implants with nanocrystalline diamond functionalized with BMP-7 promotes extracellular matrix mineralization in vitro and faster osseointegration in vivo.

The present study investigates the effect of an oxidized nanocrystalline diamond (O-NCD) coating functionalized with bone morphogenetic protein 7 (BMP-7) on human osteoblast maturation and extracellular matrix mineralization in vitro and on new bone formation in vivo. The chemical structure and the morphology of the NCD coating and the adhesion, thickness and morphology of the superimposed BMP-7 layer have also been assessed. The material analysis proved synthesis of a conformal diamond coating with a fine nanostructured morphology on the Ti6Al4V samples. The homogeneous nanostructured layer of BMP-7 on the NCD coating created by a physisorption method was confirmed by AFM. The osteogenic maturation of hFOB 1.19 cells in vitro was only slightly enhanced by the O-NCD coating alone without any increase in the mineralization of the matrix. Functionalization of the coating with BMP-7 resulted in more pronounced cell osteogenic maturation and increased extracellular matrix mineralization. Similar results were obtained in vivo from micro-CT and histological analyses of rabbit distal femurs with screws implanted for 4 or 12 weeks. While the O-NCD-coated implants alone promoted greater thickness of newly-formed bone in direct contact with the implant surface than the bare material, a further increase was induced by BMP-7. It can be therefore concluded that O-NCD coating functionalized with BMP-7 is a promising surface modification of metallic bone implants in order to improve their osseointegration.

Strong Structural and Electronic Binding of Bovine Serum Albumin to ZnO via Specific Amino Acid Residues and Zinc Atoms.

ZnO biointerfaces with serum albumin have attracted noticeable attention due to the increasing interest in developing ZnO-based materials for biomedical applications. ZnO surface morphology and chemistry are expected to play a critical role on the structural, optical, and electronic properties of albumin-ZnO complexes. Yet there are still large gaps in the understanding of these biological interfaces. Herein we comprehensively elucidate the interactions at such interfaces by using atomic force microscopy and nanoshaving experiments to determine roughness, thickness, and adhesion properties of BSA layers adsorbed on the most typical polar and non-polar ZnO single-crystal facets. These experiments are corroborated by force field (FF) and density-functional tight-binding (DFTB) calculations on ZnO-BSA interfaces. We show that BSA adsorbs on all the studied ZnO surfaces while interactions of BSA with ZnO are found to be considerably affected by the atomic surface structure of ZnO. BSA layers on the (0001‾) surface have the highest roughness and thickness, hinting at a specific upright BSA arrangement. BSA layers on (101‾0) surface have the strongest binding, which is well correlated with DFTB simulations showing atomic rearrangement and bonding between specific amino acids (AAs) and ZnO. Besides the structural properties, the ZnO interaction with these AAs also controls the charge transfer and HOMO-LUMO energy positions in the BSA-ZnO complexes. This ZnO facet-specific protein binding and related structural and electronic effects can be useful for improving the design and functionality of ZnO-based materials and devices.