Photothermal Self-Excitation of a Phase-Controlled Microcantilever for Viscosity or Viscoelasticity Sensing.

This work presents a feedback closed-loop platform to be used for viscosity or viscoelasticity sensing of Newtonian or non-Newtonian fluids. The system consists of a photothermally excited microcantilever working in a digital Phase-Locked Loop, in which the phase between the excitation signal to the cantilever and the reference demodulating signals is chosen and imposed in the loop. General analytical models to describe the frequency and amplitude of oscillation of the cantilever immersed in viscous and viscoelastic fluids are derived and validated against experiments. In particular, the sensitivity of the sensor to variations of viscosity of Newtonian fluids, or to variations of elastic/viscous modulus of non-Newtonian fluids, are studied. Interestingly, it is demonstrated the possibility of controlling the sensitivity of the system to variations of these parameters by choosing the appropriate imposed phase in the loop. A working point with maximum sensitivity can be used for real-time detection of small changes of rheological parameters with low-noise and fast-transient response. Conversely, a working point with zero sensitivity to variations of rheological parameters can be potentially used to decouple the effect of simultaneous external factors acting on the resonator.

Measuring Viscosity Using the Hysteresis of the Non-Linear Response of a Self-Excited Cantilever.

A self-oscillating microcantilever in a feedback loop comprised of a gain, a saturator, and an adjustable phase-shifter is used to measure the viscosity of Newtonian fluids. Shifting the signal of the loop with the adjustable phase-shifter causes sudden jumps in the oscillation frequency of the cantilever. The exact position of these jumps depends on whether the shift imposed by the phase-shifter is increasing or decreasing and, therefore, the self-excited cantilever exhibits a hysteretic non-linear response. This response was studied and the system modeled by a delay differential equation of motion where frequency-dependent added mass and damping terms accounted for the density and the viscosity of the medium. Experimental data were obtained for solutions with different concentrations of glycerol in water and used to validate the model. Two distinct sensing modalities were proposed for this system: the sweeping mode, where the width of the observed hysteresis depends on the viscosity of the medium, and the threshold mode, where a sudden jump of the oscillation frequency is triggered by an arbitrarily small change in the viscosity of the medium.

Microcantilever: Dynamical Response for Mass Sensing and Fluid Characterization.

A microcantilever is a suspended micro-scale beam structure supported at one end which can bend and/or vibrate when subjected to a load. Microcantilevers are one of the most fundamental miniaturized devices used in microelectromechanical systems and are ubiquitous in sensing, imaging, time reference, and biological/ biomedical applications. They are typically built using micro and nanofabrication techniques derived from the microelectronics industry and can involve microelectronics-related materials, polymeric materials, and biological materials. This work presents a comprehensive review of the rich dynamical response of a microcantilever and how it has been used for measuring the mass and rheological properties of Newtonian/non-Newtonian fluids in real time, in ever-decreasing space and time scales, and with unprecedented resolution.

Taxonomic resolution of the genus Cyanothece (Chroococcales, Cyanobacteria), with a treatment on Gloeothece and three new genera, Crocosphaera, Rippkaea, and Zehria.

The systematics of single-celled cyanobacteria represents a major challenge due to morphological convergence and application of various taxonomic concepts. The genus Cyanothece is one of the most problematic cases, as the name has been applied to oval-shaped coccoid cyanobacteria lacking sheaths with little regard to their phylogenetic position and details of morphology and ultrastructure. Hereby we analyze an extensive set of complementary genetic and phenotypic evidence to disentangle the relationships among these cyanobacteria. We provide diagnostic characters to separate the known genera Cyanothece, Gloeothece, and Aphanothece, and provide a valid description for Crocosphaera gen. nov. We describe two new genera, Rippkaea and Zehria, to characterize two distinct phylogenetic lineages outside the previously known genera. We further describe 13 new species in total including Cyanothece svehlovae, Gloeothece aequatorialis, G. aurea, G. bryophila, G. citriformis, G. reniformis, Gloeothece tonkinensis, G. verrucosa, Crocosphaera watsonii, C. subtropica, C. chwakensis, Rippkaea orientalis, and Zehria floridana to recognize the intrageneric diversity as rendered by polyphasic analysis. We discuss the close relationship of free-living cyanobacteria from the Crocosphaera lineage to nitrogen-fixing endosymbionts of marine algae. The current study includes several experimental strains (Crocosphaera and "Cyanothece") important for the study of diazotrophy and the global oceanic nitrogen cycle, and provides evidence suggesting ancestral N2 -fixing capability in the chroococcalean lineage.

Selective coupling of Whispering Gallery Modes in film coated micro-resonators.

Whispering Gallery Mode (WGM) micro-resonators like microspheres or microtoroids are typically used as high-Q cavity substrate on which a functional film coating is deposited. In order to exploit the coating properties a critical step is the efficient excitation of WGMs mainly contained inside the deposited layer. We developed a simple method able to assess whether or not these modes are selectively excited. The method is based on monitoring the thermal shift of the excited resonance, which uniquely depends on the thermo-optic coefficient and on the thermal expansion coefficient of the material in which the mode is embedded.

A Comparative Study of Phosphatidylcholine versus Phosphatidylserine-Based Solid Supported Membranes for the Preparation of Liposome-Rich Interfaces.

Solid supported membranes (SSMs) are usually formed by an hybrid octadecanethiol/phosphatidylcholine (PC) bilayer supported by a gold electrode. Recently, it was shown that phosphatidylserine (PS) in place of PC can promote a more effective accumulation of lipid vesicles on the SSM surface when Ca2+ and Mg2+ ions are present in the external environment. Here we performed a detailed comparative study of the vesicle adsorption process onto PC- and PS-SSMs by employing surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM). SPR analysis has demonstrated a higher affinity of the PS-SSM surface for the phospholipid vesicles. Both SPR and EIS measurements suggest that adsorption of lipid vesicles on the PC-SSM tends to a saturating value, whereas a continuous and progressive vesicle adsorption occurs on the PS-SSM surface following subsequent liposome additions. AFM analysis pointed out a systematic flattening of the adsorbed vesicles on the PS-SSM surface. We interpreted our results as due to the strong coordinating action of the high amount of divalent cations accumulated at the negatively charged PS-SSM surface, whereas a lower amount of cations is present on the dipolar PC-SSM surface, which can therefore adsorb only a limited number of vesicles.

Transthyretin suppresses the toxicity of oligomers formed by misfolded proteins in vitro.

Although human transthyretin (TTR) is associated with systemic amyloidoses, an anti-amyloidogenic effect that prevents Aß fibril formation in vitro and in animal models has been observed. Here we studied the ability of three different types of TTR, namely human tetramers (hTTR), mouse tetramers (muTTR) and an engineered monomer of the human protein (M-TTR), to suppress the toxicity of oligomers formed by two different amyloidogenic peptides/proteins (HypF-N and Aß42). muTTR is the most stable homotetramer, hTTR can dissociate into partially unfolded monomers, whereas M-TTR maintains a monomeric state. Preformed toxic HypF-N and Aß42 oligomers were incubated in the presence of each TTR then added to cell culture media. hTTR, and to a greater extent M-TTR, were found to protect human neuroblastoma cells and rat primary neurons against oligomer-induced toxicity, whereas muTTR had no protective effect. The thioflavin T assay and site-directed labeling experiments using pyrene ruled out disaggregation and structural reorganization within the discrete oligomers following incubation with TTRs, while confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and hTTR, particularly M-TTR. Moreover, atomic force microscopy (AFM), light scattering and turbidimetry analyses indicated that larger assemblies of oligomers are formed in the presence of M-TTR and, to a lesser extent, with hTTR. Overall, the data suggest a generic capacity of TTR to efficiently neutralize the toxicity of oligomers formed by misfolded proteins and reveal that such neutralization occurs through a mechanism of TTR-mediated assembly of protein oligomers into larger species, with an efficiency that correlates inversely with TTR tetramer stability.

Dynamical response and noise limit of a parametrically pumped microcantilever sensor in a Phase-Locked Loop.

We investigate the response of a digitally controlled and parametrically pumped microcantilever used for sensing in a Phase-Locked Loop (PLL). We develop an analytical model for its dynamical response and obtain an explicit dependence on the rheological parameters of the surrounding viscous medium. Linearization of this model allows to find improved responsivity to density variations in the case of parametric suppression. Experiments with a commercial microcantilever validate the model, but also reveal an increase of frequency noise in the PLL associated with the parametric gain and phase, which, in most cases, restricts the attainable limit of detection. The noise in open-loop is studied by measuring the random fluctuations of the noise-driven deflection of the microcantilever, and a model for the power spectral density of amplitude, phase and frequency noises is discussed and used to explain the frequency fluctuations in the closed-loop PLL. This work concludes that parametric pumping in a PLL does not improve the sensing performance in applications requiring detecting frequency shifts.

Searching for conditions to form stable protein oligomers with amyloid-like characteristics: The unexplored basic pH.

Conversion of peptides and proteins from their native states into amyloid fibrillar aggregates is the hallmark of a number of pathological conditions, including Alzheimer's disease and amyloidosis. Evidence is accumulating that soluble oligomers, as opposed to mature fibrils, mediate cellular dysfunction, ultimately leading to disease onset. In this study, we have explored the ability of alkaline pH solutions, which have remained relatively unexplored so far, to form a partially folded state of the N-terminal domain of the Escherichia coli protein HypF (HypF-N), which subsequently assembles to form stable soluble oligomers. Results showed that HypF-N unfolds at high pH via a two-state process. Characterization of the resulting alkaline-unfolded state by near- and far-UV circular dichroism, intrinsic and ANS-derived fluorescence and DLS indicated characteristics of a monomeric, premolten globule state. Interestingly, alkaline-unfolded HypF-N aggregates, at high concentration in the presence of low concentrations of TFE, into stable oligomers. These are able to bind amyloid-specific dyes, such as Congo red, ThT, and ANS, contain extensive beta-sheet structure, as detected with far-UV circular dichroism, and have a height of 2.0-3.9 nm when analysed using atomic force microscopy. This study, which complements our previous one in which morphologically, structurally, and tinctorially similar oligomers were formed at low and nearly neutral pH values by the same protein, offers opportunities to explore the fine differences existing in the mechanism of formation of these species under different conditions, in their precise molecular structure and in their ability to cause cellular dysfunction.

Characterization of ordered aggregates of cerato-platanin and their involvement in fungus-host interactions.

BACKGROUND: The "cerato-platanin family" consists of fungal-secreted proteins that are involved in various stages of the host-fungus interaction and act as phytotoxins, elicitors of defense responses and allergens. Cerato-platanin (CP) is a moderately hydrophobic protein secreted and localized in the cell wall of Ceratocystis platani, the causal agent of a severe disease of Platanus. These properties make CP like the hydrophobins: these are self-assembling proteins that form a surface coating which is involved in the formation of aerial hyphae and in adherence to surfaces. METHODS: CP aggregation was monitored by ThT, circular dichroism, and AFM. The eliciting activity of CP aggregates was assayed on leaves and cells. RESULTS: The CP self-assembles forming amyloid-like aggregates via a nucleated growth mechanism which is joined up with a cleavage of the N-terminus. The ovoidal shape and the lack of a clear transition toward an all-beta structure distinguish these aggregates from typical amyloid fibrils. Moreover, CP aggregates interact with hydrophobic surfaces and enhance the hypersensitive response of Platanus. CONCLUSION AND GENERAL SIGNIFICANCE: CP forms "ordered aggregates" for which the soluble prefibrillar structures are the end point of the aggregation process, and do not evolve to insoluble fibrils. An involvement in host-microbe interaction is also suggested.

Progressive pearl necklace collapse mechanism for cerato-ulmin aggregation film.

Cerato-ulmin (CU) is a fungal toxin class II hydrophobin, involved in Dutch elm disease. The formation of hydrophobin films at the air-water interface is a key mechanism which plays a role of paramount importance at different stages of the fungal development. We present a study on the precursor stages of growth towards the self-assembly aggregation film of CU. Atomic force microscopy images of CU dropped on mica substrates indicate that the system self-organizes in almost one-dimensional pearl-necklace-like chains, which subsequently collapse and possibly merge to form extended and rather compact planar films. We propose and verify a simple model to describe the self-aggregation mechanism in terms of progressive thickening of the pearl chains due to the successive merging and collapse of the elementary constitutive units.

The In Vitro Anti-amyloidogenic Activity of the Mediterranean Red Seaweed Halopithys Incurva.

Neurodegenerative diseases are generally characterized by the presence of neurotoxic amyloid aggregates underlying progressive neuronal death. Since ancient times, natural compounds have been used as curative agents for human health. Amyloid research is constantly looking for safe natural molecules capable of blocking toxic amyloid aggregates' formation. From the marine environment, seaweeds are recognized as rich reservoirs of molecules with multiple bioactivities, including the anti-amyloidogenic activity. Here, hydroalcoholic extracts of two seasonal samples of the Mediterranean red seaweed Halophytis incurva (HIEs) were characterized by the HPLC-DAD-MS analysis. The H. incurva anti-amyloidogenic role was explored by incubating both HIEs with hen egg white lysozyme (HEWL), a well-known protein model widely used in amyloid aggregation experiments. The aggregation kinetics and morphological analysis of amyloid aggregates were performed by ThT and AFM analysis, respectively, while their cytotoxicity on SH-SY5Y human neuroblastoma cells was examined by MTT assay. HIEs showed a different efficacy, probably dependent on their metabolic composition, both in inhibiting amyloid fibrillation and in obtaining short and less toxic pre-fibrillary aggregates. Overall, this work sheds light, for the first time, on a Mediterranean red seaweed as a promising renewable resource of bioactive compounds, potentially useful in preventing the formation of toxic amyloid aggregates.

Fiber-cap biosensors for SERS analysis of liquid samples.

Optical detection techniques based on surface enhanced Raman spectroscopy (SERS) are a powerful tool for biosensing applications. Meanwhile, due to technological advances, different approaches have been investigated to integrate SERS substrates on the tip of optical fibres for molecular probing in liquids. To further demonstrate the perspectives offered by SERS-on-fiber technology for diagnostic purposes, in this study, novel cap-shaped SERS sensors for reversible coupling with customized multimodal probes were prototyped via low-cost polymer casting of polydimethylsiloxane (PDMS) and further assembly of gold nanoparticles (Au NPs) of varied sizes and shapes. To demonstrate the feasibility of liquid sensing with cap sensors using backside illumination and detection, the spectra of rhodamine were acquired by coupling the caps with the fiber. As expected by UV-vis, the highest SERS efficiency was observed for NP-decorated substrates with plasmonic properties in resonance with the irradiation wavelength. Then, SERS biosensors for the specific detection of amyloid-ß (Aß) neurotoxic biomarkers were realized by covalent grafting of Aß antibodies. As attested by fluorescence images and SERS measurements, the biosensors successfully exhibited enhanced Aß affinity compared to the bare sensors without ligands. Finally, these versatile (bio)sensors are a powerful tool to transform any milli-sized fibers into functional (bio)sensing platforms with plasmonic and biochemical properties tailored for specific applications.

New proteins orthologous to cerato-platanin in various Ceratocystis species and the purification and characterization of cerato-populin from Ceratocystis populicola.

Natural variants of cerato-platanin (CP), a pathogen associated molecular pattern (PAMP) protein produced by Ceratocystis platani (the causal agent of the plane canker stain), have been found to be produced by other four species of the genus Ceratocystis, including five clones of Ceratocystis fimbriata isolated from different hosts. All these fungal strains were known to be pathogenic to plants with considerable importance in agriculture, forestry, and as ornamental plants. The putative premature proteins were deduced on the basis of the nucleotide sequence of genes orthologous to the cp gene of C. platani; the deduced premature proteins of Ceratocystis populicola and Ceratocystis variospora reduced the total identity of all the others from 87.3% to 60.3%. Cerato-populin (Pop1), the CP-orthologous protein produced by C. populicola, was purified and characterized. Pop1 was a well-structured alpha/beta protein with a different percentage of the alpha-helix than CP, and it self-assembled in vitro in ordered aggregates. Moreover, Pop1 behaved as PAMP, since it stimulated poplar leaf tissues to activate defence responses able to reduce consistently the C. populicola growth.

Atomic force microscopy and transmission electron microscopy analyses of low-temperature laser welding of the cornea.

Low-temperature laser welding of the cornea is a technique used to facilitate the closure of corneal cuts. The procedure consists of staining the wound with a chromophore (indocyanine green), followed by continuous wave irradiation with an 810 nm diode laser operated at low power densities (12-16 W/cm(2)), which induces local heating in the 55-65 degrees C range. In this study, we aimed to investigate the ultrastructural modifications in the extracellular matrix following laser welding of corneal wounds by means of atomic force microscopy and transmission electron microscopy. The results evidenced marked disorganization of the normal fibrillar assembly, although collagen appeared not to be denatured under the operating conditions we employed. The mechanism of low-temperature laser welding may be related to some structural modifications of the nonfibrillar extracellular components of the corneal stroma.

Self-Assembled Nanocrystals of Polycyclic Aromatic Hydrocarbons Show Photostable Single-Photon Emission.

Quantum technologies could largely benefit from the control of quantum emitters in sub-micrometric size crystals. These are naturally prone to integration in hybrid devices, including heterostructures and complex photonic devices. Currently available quantum emitters in nanocrystals suffer from spectral instability, preventing their use as single-photon sources for most quantum optics operations. In this work we report on the performances of single-photon emission from organic nanocrystals (average size of hundreds of nm), made of anthracene (Ac) and doped with dibenzoterrylene (DBT) molecules. The source has hours-long photostability with respect to frequency and intensity, both at room and at cryogenic temperature. When cooled to 3 K, the 00-zero phonon line shows linewidth values (50 MHz) close to the lifetime limit. Such optical properties in a nanocrystalline environment recommend the proposed organic nanocrystals as single-photon sources for integrated photonic quantum technologies.

Catechol-Containing Hydroxylated Biomimetic 4-Thiaflavanes as Inhibitors of Amyloid Aggregation.

The study of compounds able to interfere in various ways with amyloid aggregation is of paramount importance in amyloid research. Molecules characterized by a 4-thiaflavane skeleton have received great attention in chemical, medicinal, and pharmaceutical research. Such molecules, especially polyhydroxylated 4-thiaflavanes, can be considered as structural mimickers of several natural polyphenols that have been previously demonstrated to bind and impair amyloid fibril formation. In this work, we tested five different 4-thiaflavanes on the hen egg-white lysozyme (HEWL) amyloid model for their potential anti-amyloid properties. By combining a thioflavin T assay, atomic force microscopy, and a cell toxicity assay, we demonstrated that such compounds can impair the formation of high-order amyloid aggregates and mature fibrils. Despite this, the tested 4-thiaflavanes, although non-toxic per se, are not able to prevent amyloid toxicity on human neuroblastoma cells. Rather, they proved to block early aggregates in a stable, toxic conformation. Accordingly, 4-thiaflavanes can be proposed for further studies aimed at identifying blocking agents for the study of toxicity mechanisms of amyloid aggregation.

Controlled graphene oxide assembly on silver nanocube monolayers for SERS detection: dependence on nanocube packing procedure.

Hybrid graphene oxide/silver nanocubes (GO/AgNCs) arrays for surface-enhanced Raman spectroscopy (SERS) applications were prepared by means of two procedures differing for the method used in the assembly of the silver nanocubes onto the surface: Langmuir-Blodgett (LB) transfer and direct sequential physisorption of silver nanocubes (AgNCs). Adsorption of graphene oxide (GO) flakes on the AgNC assemblies obtained with both procedures was monitored by quartz crystal microbalance (QCM) technique as a function of GO bulk concentration. The experiment provided values of the adsorbed GO mass on the AgNC array and the GO saturation limit as well as the thickness and the viscoelastic properties of the GO film. Atomic force microscopy (AFM) measurements of the resulting samples revealed that a similar surface coverage was achieved with both procedures but with a different distribution of silver nanoparticles. In the GO covered LB film, the AgNC distribution is characterized by densely packed regions alternating with empty surface areas. On the other hand, AgNCs are more homogeneously dispersed over the entire sensor surface when the nanocubes spontaneously adsorb from solution. In this case, the assembly results in less-packed silver nanostructures with higher inter-cube distance. For the two assembled substrates, AFM of silver nanocubes layers fully covered with GO revealed the presence of a homogeneous, flexible and smooth GO sheet folding over the silver nanocubes and extending onto the bare surface. Preliminary SERS experiments on adenine showed a higher SERS enhancement factor for GO on Langmuir-Blodgett films of AgNCs with respect to bare AgNC systems. Conversely, poor SERS enhancement for adenine resulted for GO-covered AgNCs obtained by spontaneous adsorption. This indicated that the assembly and packing of AgNCs obtained in this way, although more homogeneous over the substrate surface, is not as effective for SERS analysis.

Controlled Veiling of Silver Nanocubes with Graphene Oxide for Improved Surface-Enhanced Raman Scattering Detection.

Hybrid graphene oxide (GO)/metal nanocomposites have been recently proposed as novel surface-enhanced Raman scattering (SERS) substrates. Despite an increasing interest in these systems, standardization in their fabrication process is still lacking but urgently required to support their use for real-life applications. In this work we investigate how the assembly of GO should be conducted to control adsorption geometry and optical properties at the interface with plasmonic nanostructures as monolayer assemblies of silver nanocubes, by tuning main experimental parameters including GO concentration and self-assembly time. We finally identified the experimental conditions for building up a close-fitting soft dressing of the plasmonic surface, which shows optimal characteristics for flexible and reliable SERS detection.

Mechanisms for the inhibition of amyloid aggregation by small ligands.

The formation of amyloid aggregates is the hallmark of systemic and neurodegenerative disorders, also known as amyloidoses. Many proteins have been found to aggregate into amyloid-like fibrils and this process is recognized as a general tendency of polypeptides. Lysozyme, an antibacterial protein, is a well-studied model since it is associated in human with systemic amyloidosis and that is widely available from chicken eggs (HEWL, hen egg white lysozyme). In the present study we investigated the mechanism of interaction of aggregating HEWL with rosmarinic acid and resveratrol, that we verified to be effective and ineffective, respectively, in inhibiting aggregate formation. We used a multidisciplinary strategy to characterize such effects, combining biochemical and biophysical methods with molecular dynamics (MD) simulations on the HEWL peptide 49-64 to gain insights into the mechanisms and energy variations associated to amyloid formation and inhibition. MD revealed that neither resveratrol nor rosmarinic acid were able to compete with the initial formation of the ß-sheet structure. We then tested the association of two ß-sheets, representing the model of an amyloid core structure. MD showed that rosmarinic acid displayed an interaction energy and a contact map comparable to that of sheet pairings. On the contrary, resveratrol association energy was found to be much lower and its contact map largely different than that of sheet pairings. The overall characterization elucidated a possible mechanism explaining why, in this model, resveratrol is inactive in blocking fibril formation, whereas rosmarinic acid is instead a powerful inhibitor.