Fluorimetric Cell Analysis with 9-Aryl-Substituted Berberine Derivatives as DNA-Targeting Fluorescent Probes.

DNA-sensitive fluorescent light-up probes based on berberine are presented. This biogenic fluorophore was chosen as central unit to use its potential biocompatibility and its DNA-binding properties. To provide predictable fluorescence quenching in aqueous solution and a fluorescence light-up effect upon DNA binding, aryl substituents were attached at the 9-position by Suzuki-Miyaura coupling reactions. The 9-arylberberine derivatives have a very low fluorescence quantum yield (Φfl =<0.02), which is caused by the radiationless deactivation of the excited state by torsional relaxation about the biaryl axis. In addition, these berberine derivatives intercalate into DNA with high affinity (Kb =2.0-22×104  M-1 ). Except for the nitrophenyl- and hydroxyphenyl-substituted derivatives, all tested compounds exhibited a pronounced fluorescence light-up effect upon association with DNA, because the deactivation of the excited-state by torsional relaxation is suppressed in the DNA binding site. Most notably, it was shown exemplarily with the 9-(4-methoxyphenyl)- and the 9-(6-methoxynaphthyl)-substituted derivatives that these properties are suited for fluorimetric cell analysis. In particular, these probes generated distinct staining patterns in eukaryotic cells (NIH 3T3 mouse fibroblasts), which enabled the identification of nuclear substructures, most likely heterochromatin or nucleoli, respectively.

Investigation of the Orientation and Assembly of Functionalized Microcubes at the Oil-Water Interface.

The dependence of the preferred orientation of polystyrene microcubes on surface hydrophobicity at the water/hexadecane interface is reported. Similar to the water/air interfaces, the microcubes were shown to reside at the water/hexadecane interface with three distinct orientations: face-up, edge-up, and vertex-up. Concomitantly, ordered aggregates with flat plate, tilted linear, and close-packed hexagonal structures were formed, driven by capillary force. With increasing the hydrophobicity of five sides of the cubes, the preferential microcube orientation at the water/hexadecane interface changed sequentially from face-up to edge-up, to vertex-up, then back to edge-up, and to face-up. This dependence of the preferential microcube orientation on surface hydrophobicity at the water/hexadecane interface differs from that observed at the water/air interface, where the preferential orientation changed only from face-up to edge-up, then to vertex-up, as surface hydrophobicity increased. In addition, preformed microcube assemblies at the water/air interface could be dynamically reconfigured by replacing the air phase with hexadecane under stirring.

Effect of Particle Size on the Orientation and Order of Assemblies of Functionalized Microscale Cubes Formed at the Water/Air Interface.

The impact of the particle size and wettability on the orientation and order of assemblies obtained by self-organization of functionalized microscale polystyrene cubes at the water/air interface is reported. An increase in the hydrophobicity of 10- and 5-µm-sized self-assembled monolayer-functionalized polystyrene cubes, as assessed by independent water contact angle measurements, led to a change of the preferred orientation of the assembled cubes at the water/air interface from face-up to edge-up and further to vertex-up, irrespective of microcube size. This tendency is consistent with our previous studies with 30-µm-sized cubes. However, the transitions among these orientations and the capillary force-induced structures, which change from flat plate to tilted linear and further to close-packed hexagonal arrangements, were observed to shift to larger contact angles for smaller cube size. Likewise, the order of the formed aggregates decreased significantly with decreasing cube size, which is tentatively attributed to the small ratio of inertial force to capillary force for smaller cubes in disordered aggregates, which results in more difficulties to reorient in the stirring process. Experiments with small fractions of larger cubes added to the water/air interface increased the order of smaller homo-aggregates to values similar to neat 30 µm cube assemblies. Hence, collisions of larger cubes or aggregates are shown to play a decisive role in breaking metastable structures to approach a global energy minimum assembly.

Silver nanoparticles adversely affect the swimming behavior of European Whitefish (Coregonus lavaretus) larvae within the low µg/L range.

The aim of this study was to determine the effects of silver nanoparticles (AgNPs; speciation: NM-300 K) in the lab on the behavior of larvae in European Whitefish (Coregonus lavaretus), a relevant model species for temperate aquatic environments during alternating light and darkness phases. The behavioral parameters measured included activity, turning rate, and distance moved. C. lavaretus were exposed to AgNP at nominal concentrations of 0, 5, 15, 45, 135, or 405 µg/L (n = 33, each) and behavior was recorded using a custom-built tracking system equipped with light sources that reliably simulate light and darkness. The observed behavior was analyzed using generalized linear mixed models, which enabled reliable detection of AgNP-related movement patterns at 10-fold higher sensitivity compared to recently reported standard toxicological studies. Exposure to 45 µg/L AgNPs significantly resulted in hyperactive response patterns for both activity and turning rates after an illumination change from light to darkness suggesting that exposure to this compound triggered escape mechanisms and disorientation-like behaviors in C. lavaretus fish larvae. Even at 5 µg/L AgNPs some behavioral effects were detected, but further tests are required to assess their ecological relevance. Further, the behavior of fish larvae exposed to 135 µg/L AgNPs was comparable to the control for all test parameters, suggesting a triphasic dose response pattern. Data demonstrated the potential of combining generalized linear mixed models with behavioral investigations to detect adverse effects on aquatic species that might be overlooked using standard toxicological tests.

Incubation media modify silver nanoparticle toxicity for whitefish (Coregonus lavaretus) and roach (Rutilus rutilus) embryos.

Toxicological studies were performed to examine silver nanoparticle (AgNP, size: 14.4 ± 2.5 nm) transformation within three different test media and consequent effects on embryos of whitefish (Coregonus lavaretus) and roach (Rutilus rutilus). The test media, namely ASTM very hard water, ISO standard dilution medium, and natural lake water differed predominantly in ionic strength. Total silver was determined using inductively coupled plasma mass spectrometry (ICP-MS), while AgNPs were characterized by transmission electron microscopy and single particle ICP-MS. Silver species distributions were estimated via thermodynamic speciation calculations. Data demonstrated that increased AgNP dissolution accompanied by decreasing ionic strength of the test medium did not occur as noted in other studies. Further, other physicochemical parameters including AgNP size and metallic species distribution did not markedly affect AgNP-induced toxicity. Irrespective of the test medium, C. lavaretus were more sensitive to AgNP exposure (median lethal concentration after 8 weeks: 0.51-0.73 mg/L) compared to R. rutilus, where adverse effects were only observed at 5 mg/L in natural lake water. In addition, AgNP-induced toxicity was lower in the two standard test media compared to natural lake water. Currently, there are no apparent studies assessing simultaneously the sensitivity of C. lavaretus and R. rutilus to AgNP exposure. Therefore, the aim of this study was to (1) investigate AgNP-induced toxicity in C. lavaretus and R. rutilus cohabiting in the same aquatic environment and (2) the role played by test media in the observed effects of AgNPs on these aquatic species.

Restoring Endogenous Repair Mechanisms to Heal Chronic Wounds with a Multifunctional Wound Dressing.

Current treatment of chronic wounds has been critically limited by various factors, including bacterial infection, biofilm formation, impaired angiogenesis, and prolonged inflammation. Addressing these challenges, we developed a multifunctional wound dressing-based three-pronged approach for accelerating wound healing. The multifunctional wound dressing, composed of nanofibers, functional nanoparticles, natural biopolymers, and selected protein and peptide, can target multiple endogenous repair mechanisms and represents a promising alternative to current wound healing products.

In Situ Study of Layer-by-Layer Polyelectrolyte Deposition in Nanopores of Anodic Aluminum Oxide by Reflectometric Interference Spectroscopy.

The modification of cylindrical anodic aluminum oxide (AAO) nanopores by alternating layer-by-layer (LBL) deposition of poly(sodium-4-styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) was studied in situ by reflectometric interference spectroscopy (RIfS). In particular, the kinetics of polyelectrolyte deposition inside the pores with a diameter of 37 ± 3 nm and a length of 3.7 ± 0.3 µm were unraveled, and potential differences in the LBL multilayer growth compared to flat silicon substrates as well as the effect of different ionic strengths and different types of ions were investigated. RIfS measures the effective optical thicknesses, which is-for a constant pore length-proportional to the effective refractive index of the AAO sample, from which, in turn, the deposited mass of the polymer or the corresponding layer thickness can be estimated. Compared to the multilayer growth by the LBL deposition on the flat aminosilane-primed silicon wafers, which was assessed by spectroscopic ellipsometry, the thickness increment per deposited bilayer, as well as the dependence of this increment on the ionic strength (0.01-0.15) and the counterion type (Na+ vs Ca2+) inside the aminosilane-primed nanopores, was for the first bilayers to within the experimental error identical. For thicker multilayers, the pore diameter became smaller, which led to reduced thickness increments and eventually virtually completely filled the pores. The observed kinetics is consistent with the mass-transport-limited adsorption of the polyelectrolyte to the charged surface according to a Langmuir isotherm with a negligible desorption rate. In addition to fundamental insights into the buildup of polyelectrolyte multilayers inside the AAO nanopores, our results highlight the sensitivity of RIfS and its use as an analytical tool for probing processes inside the nanopores and for the development of biosensors.

Poly(diethylene glycol methylether methacrylate) Brush-Functionalized Anodic Alumina Nanopores: Curvature-Dependent Polymerization Kinetics and Nanopore Filling.

We report on the synthesis and characterization of poly(diethylene glycol methylether methacrylate) (PDEGMA) brushes by surface-initiated atom transfer radical polymerization inside ordered cylindrical nanopores of anodic aluminum oxide with different pore radii between 20 and 185 nm. In particular, the dependence of polymerization kinetics and the degree of pore filling on the interfacial curvature were analyzed. On the basis of field emission scanning electron microscopy data and thermal gravimetric analysis (TGA), it was concluded that the polymerization rate was faster at the pore orifice compared to the pore interior and also as compared to the analogous reaction carried out on flat aluminum oxide substrates. The apparent steady-state polymerization rate near the orifice increased with decreasing pore size. Likewise, the overall apparent polymerization rate estimated from TGA data indicated stronger confinement for pores with increased curvature as well as increased mass transport limitations due to the blockage of the pore orifice. Only for pores with a diameter to length ratio of ∼1, PDEGMA brushes were concluded to grow uniformly with constant thickness. However, because of mass transport limitations in longer pores, incomplete pore filling was observed, which leads presumably to a PDEGMA gradient brush. This study contributes to a better understanding of polymer brush-functionalized nanopores and the impact of confinement, in which the control of polymer brush thickness together with grafting density along the nanopores is key for applications of PDEGMA brushes confined inside nanopores.

Investigation of the Fate of Silver and Titanium Dioxide Nanoparticles in Model Wastewater Effluents via Selected Area Electron Diffraction.

The increasing use of manufactured nanomaterials (MNMs) and their inevitable release into the environment, especially via wastewater treatment plants (WWTPs), poses a potential threat for aquatic organisms. The characterization of MNMs with analytical tools to comprehend their fate and effect on the ecosystem is hence of great importance for environmental risk assessment. We herein report, for the first time, the investigation of physicochemical transformation processes during artificial wastewater treatment of silver (Ag-NPs) and titanium dioxide nanoparticles (TiO2-NPs) via selected area electron diffraction (SAED). TiO2-NPs with an anatase/rutile ratio of ∼80/20 were found to not undergo any physicochemical transformation, as shown via previous energy-dispersive X-ray analysis (EDX) elemental mapping and crystal structure analysis via SAED. In contrast, Ag-NPs were colocalized with substantial amounts of sulfur (Ag/S ratio of 1.9), indicating the formation of Ag2S. SAED ultimately proved the complete transformation of face-centered cubic (fcc) Ag-NPs into monoclinic Ag2S-NPs. The size distribution of both nanomaterials remained virtually unchanged. Our investigations show that cloud point extraction of NPs and their subsequent crystal structure analysis via SAED is another valuable approach toward the comprehensive investigation of wastewater-borne MNMs. However, the extraction procedure needs optimization for environmentally low NP concentrations.

Colorimetric and Fluorimetric DNA Detection with a Hydroxystyryl-Quinolizinium Photoacid and Its Application for Cell Imaging.

The combination of styryl dye properties with the acidity and strong photoacidity of the 2,2'-[(1''-hydroxy-4''-methyl-(E)-2'',6''-phenylene)]-bisquinolizinium enables the detection of DNA by distinct absorption and emission color changes and the fluorimetric detection of DNA in cells with epifluorescence and confocal fluorescence microscopy.

Control of Orientation, Formation of Ordered Structures, and Self-Sorting of Surface-Functionalized Microcubes at the Air-Water Interface.

The dependence of the orientation of microscale PS cubes, which are surface functionalized on only five faces, at the water/air interface and the ordered aggregates formed by capillary force assembly are reported. Depending on the wettability of the faces, the cubes were shown to adopt a preferred orientation that changes with decreasing wettability from face up to edge up and further to vertex up. Concomitantly, stable aggregates with different structures were formed by capillary force self-assembly. The unmodified bottom face of the cubes was localized by fluorescence labeling. Finally, self-sorting of differently surface functionalized microcubes was realized for the first time, due to the stronger capillary interactions of quadrupole-quadrupole and hexapole-hexapole interactions compared to quadrupole-hexapole interaction.

Reconfigurable Microcube Assemblies at the Liquid/Air Interface: The Impact of Surface Tension on Orientation and Capillary-Force-Interaction-Driven Assembly.

The systematic investigation of the dependence of the orientation and capillary interaction of hydrophobized polystyrene microcubes at the liquid/air interface on the surface tension of the aqueous subphase is reported. By decreasing the subphase surface tension, the preferential orientation of the cubes was observed to change independent of the surfactant type from the vertex up to the edge up and finally to the face up. Concomitantly, the structure of the aggregates obtained by cube assembly was observed to change from a close-packed hexagonal to tilted linear and finally to flat plate. In particular, the preferential orientation of the cubes was virtually independent of the surfactant charge at a constant surface tension. In addition, reconfigurable microcube assemblies at the liquid/air interface, which respond to the surface tension of the subphase, were observed for the first time. The dynamic reconfigurability of preformed microcube aggregates induced by adding surfactant to the subphase may open new pathways to dynamic assemblies at liquid/air interfaces, which may be interesting, e.g., for sensing applications.

Propagation and Purification of Human Induced Pluripotent Stem Cells with Selective Homopolymer Release Surfaces.

The selective detachment of undifferentiated human induced pluripotent stem (iPS) cells from a thermal release coating, fabricated from a tailored poly(di(ethylene glycol) methyl ether methacrylate) (PDEGMA) homopolymer layer on gold, is reported. By exploiting the mild, thermally triggered release of iPS cell colonies in the absence of any releasing reagent, pluripotent iPS cells are shown to be selectively separated from spontaneously differentiated cells. The maintained pluripotency and high cell viability of detached and reseeded iPS cell colonies were confirmed and suggest the feasibility of a generally applicable platform approach for cell separation and purification in the context of iPS cell culture, differentiation of pathologically altered cells and normal cells, as well as isolation of different cell types derived from certain tissues, for example, from biopsies.

Tailored Combinatorial Microcompartments through the Self-Organization of Microobjects: Assembly, Characterization, and Cell Studies.

A new concept enables the generation of cell microenvironments by microobject assembly at an water/air interface. As the orientation of 30 µm sized polymer cubes and their capillary force assembly are controlled by the surface wettability, which in turn can be modulated by coating the initially exposed surfaces with gold and self-assembled monolayers, unique niches in closely packed arrays of cubes with vertex up orientation can be realized. The random assembly of distinctly different cubes, prefunctionalized or surface-structured exclusively on their top surface, facilitates the parallel generation of different microenvironments in a combinatorial manner, which paves the way to future systematic structure-property relationship studies with cells.

Probing of local polarity in poly(methyl methacrylate) with the charge transfer transition in Nile red.

The permittivity of polymers and its spatial distribution play a crucial role in the behavior of thin films, such as those used, e.g., as sensor coatings. In an attempt to develop a conclusive approach to determine these quantities, the polarity of the model polymer poly(methyl methacrylate) (PMMA) in 600 nm thin films on a glass support was probed by the energy of the charge transfer transition in the oxazine dye Nile red (NR) at 25 °C. The absorption and fluorescence spectra of NR were observed to shift to the red with increasing solvent polarity, because of the intramolecular charge transfer character of the optical transition. New types of solvatochromic plots of emission frequency against absorption frequency and vice versa afforded the Onsager radius-free estimation of the ground and excited states dipole moment ratio. With this approach the values of these dipole moments of 11.97 D and 18.30-19.16 D, respectively, were obtained for NR. An effective local dielectric constant of 5.9-8.3 for PMMA thin films was calculated from the solvatochromic plot and the fluorescence maximum of NR observed in the PMMA films. The fluorescence band of NR in the rigid PMMA films shifted to the red by 130 cm-1 with increasing excitation wavelength from 470 to 540 nm, while in a series of liquids the position of the emission maximum of NR remained constant within same range of the excitation wavelength. It is concluded that the fluorescence spectrum of NR in PMMA undergoes inhomogeneous broadening due to different surroundings of NR molecules in the ground state and slow sub-glass transition (T g) relaxations in PMMA.

3D 3C-SiC/Graphene Hybrid Nanolaminate Films for High-Performance Supercapacitors.

High-performance supercapacitors feature big and stable capacitances and high power and energy densities. To fabricate high-performance supercapacitors, 3D 3C-SiC/graphene hybrid nanolaminate films are grown via a microwave plasma-assisted chemical vapor deposition technique. Such films consist of 3D alternating structures of vertically aligned 3C-SiC and graphene layers, leading to high surface areas and excellent conductivity. They are further applied as the capacitor electrodes to construct electrical double layer capacitors (EDLCs) and pseudocapacitors (PCs) in both aqueous and organic solutions. The capacitance for an EDLC in aqueous solutions is up to 549.9 µF cm-2 , more than 100 times higher than that of an epitaxial 3C-SiC film. In organic solutions, it is 297.3 µF cm-2 . The pseudocapacitance in redox-active species (0.05 Fe(CN)6 3-/4- ) contained aqueous solutions is as high as 62.2 mF cm-2 . The capacitance remains at 98% of the initial value after 2500 charging/discharging cycles, indicating excellent cyclic stability. In redox-active species (0.01 m ferrocene) contained organic solutions, it is 16.6 mF cm-2 . Energy and power densities of a PC in aqueous solution are 11.6 W h kg-1 and 5.1 kW kg-1 , respectively. These vertically aligned 3C-SiC/graphene hybrid nanolaminate films are thus promising electrode materials for energy storage applications.

Thickness-Encoded Micropatterns in One-Component Thermoresponsive Polymer Brushes for Culture and Triggered Release of Pancreatic Tumor Cell Monolayers and Spheroids.

Fabrication, characterization, and application of micropatterned one-component poly(di(ethylene glycol)methyl ether methacrylate) (PDEGMA) brushes for monolayer cell and spheroid culture and temperature-triggered release are reported. Micropatterns of various shapes and sizes were designed to possess a unique functionality imparted by thermoresponsive thin PDEGMA patches, which are cell adhesive at 37 °C, embedded in a much thicker cell-resistant PDEGMA matrix that does not exhibit measurable thermoresponsive properties. Depending on the cell seeding density, PaTu 8988t human pancreatic tumor cells or spheroids were cultured area-selectively, confined by the 40 ± 4 nm thick passivating PDEGMA matrix, and could be released on demand by a mild thermally triggered brush swelling in the 5 ± 1 nm thin regions. As shown by surface plasmon resonance (SPR) measurements, in contrast to the thinner brushes, the thicker brushes exhibited virtually no fibronectin adhesive properties at 37 °C, whereas at 25 °C, both areas showed similar protein resistant behavior. The quasi-2D thickness-encoded micropatterns were shown to be useful templates for the growth of 3D multicellular aggregates. Thermally induced release after 5 days of incubation afforded 3D cell spheroids comprising up to 99% viable cells demonstrating that the system can be used as a 3D spheroid in vitro model for basic tumor research and anticancer drug screenings.

Fluorimetric Detection of G-Quadruplex DNA in Solution and Adsorbed on Surfaces with a Selective Trinuclear Cyanine Dye.

Quadruplex DNA, which is a relevant target for anticancer therapies, may alter its conformation because of interactions with interfaces. In pursuit of a versatile methodology to probe adsorption-induced conformational changes, the interaction between a fluorescent [2.2.2]heptamethinecyanine dye and quadruplex DNA (G4-DNA) was studied in solution and on surfaces. In solution, the cyanine dye exhibits a strong light-up effect upon the association with G4-DNA without interference from double-stranded DNA. In addition, a terminal π-stacking as a binding mode between the cyanine dye and G4-DNA is concluded using NMR spectroscopy. To unravel the effects of adsorption on the conformation of quadruplex-DNA, G4-DNA, and double-stranded and single-stranded DNA were adsorbed to positively charged poly(allylamine) hydrochloride (PAH) surfaces, both in planar and in constrained 55 nm diameter aluminum oxide nanopore formats. All DNA forms showed a very strong affinity to the PAH surfaces as shown by surface plasmon resonance and reflectometric interference spectroscopy. The significant increase of the fluorescence emission intensity of the cyanine light-up probe observed exclusively for surface immobilized G4-DNA affords evidence for the adsorption of G4-DNA on PAH with retained quadruplex conformation.

Three-Dimensional Microstructured Poly(vinyl alcohol) Hydrogel Platform for the Controlled Formation of Multicellular Cell Spheroids.

Three-dimensional (3D) multicellular cell spheroids (MCSs) are excellent in vitro cell models, in which, e.g., the in vivo cell-cell interaction processes are much better mimicked than in conventional two-dimensional (2D) cell layers. However, the difficulties in the generation of well-defined MCSs with controlled size severely limit their application. Herein, low-adhesive poly(vinyl alcohol) (PVA) hydrogels structured with inverted pyramid-shaped microwells were used to guide the aggregation of cells into MCSs. The cells settling down into the microwells by gravity accumulated at the central tip of the wells and then gradually grew into spheroids. The size of cell spheroids can be straightforwardly controlled by the culture time and initially seeded cell number. The MCSs generated in a parallel microarray format were further used for drug testing. Our results suggest in agreement with complementary literature data that the cell culture format plays a critical role in the cellular response to drugs, and also confirms that spheroids possess a much higher drug resistance than cells in 2D layers. This novel microstructured PVA hydrogel is expected to offer a potential platform for the facile preparation of spheroids for various applications in the biomedical field.

Rapid determination of binding parameters of chitin binding domains using chitin-coated quartz crystal microbalance sensor chips.

Chitin present in fungal cell walls has been considered as a diagnostic polymer for the detection of fungal infections. Chitin staining can be achieved with different dyes such as Calcofluor white or Congo red, but these methods have not entered into clinical routine diagnosis due to problems with sensitivity and specificity. More accurate detection can be achieved using chitin binding domains (CBDs) from a large variety of naturally occurring proteins that specifically interact with chitin. The chitin binding properties of most of these proteins have not yet been determined, because chitin is an insoluble fibrillar material rendering accurate determination of chitin binding kinetics challenging. Here we report a quartz crystal microbalance with dissipation monitoring (QCM-D) based method to determine binding constants of CBDs on chitin-coated gold surfaces. For this purpose, chitin was trimethylsilylated and coated onto the sensor chips. After desilylation, regular fibril-like structures with a typical center-to-center spacing of 85 nm were observed by atomic force microscopy. Using different experimental conditions and data evaluation methods for QCM-D measurements, we determined kon and koff and calculated the KD values for binding of a recombinant CBD from Bacillus circulans chitinase A1. Depending on the evaluation method, the KD values ranged between 0.6 and 2.5 µM. The obtained KD values were in good agreement with those measured for other bacterial CBDs usually ranging between 1 to 10 µM. Hence, we propose that the experimental approach developed in this study can be applied to determine yet unknown binding affinities of various CBDs from different origin.