Glial-fibrillary-acidic-protein (GFAP) biomarker detection in serum-matrix: Functionalization strategies and detection by an ultra-high-frequency surface-acoustic-wave (UHF-SAW) lab-on-chip.

Glial-fibrillary-acidic-protein (GFAP) has recently drawn significant attention from the clinical environment as a promising biomarker. The pathologies which can be linked to the presence of GFAP in blood severely affect the human central nervous system. These pathologies are glioblastoma multiforme (GBM), traumatic brain injuries (TBIs), multiple sclerosis (MS), intracerebral hemorrhage (ICH), and neuromyelitis optica (NMO). Here, we develop three different detection strategies for GFAP, among the most popular in the biosensing field and never examined side by side within the experimental frame. We compare their capability of detecting GFAP in a clean-buffer and serum-matrix by using gold-coated quartz-crystal-microbalance (QCM) sensors. All the three detection strategies are based on antibodies, and each of them focuses on a key aspect of the biosensing process. The first is based on a polyethylene glycol (PEG) chain for antifouling, the second on a protein-G linker for controlling antibody-orientation, and the third on antibody-splitting and direct surface immobilization for high-surface coverage. Then, we select the best-performing protocol and validate its detection performance with an ultra-high-frequency (UHF) surface-acoustic-wave (SAW) based lab-on-chip (LoC). GFAP successful detection is demonstrated in a clean-buffer and serum-matrix at a concentration of 35 pM. This GFAP level is compatible with clinical diagnostics. This result suggests the use of our technology for the realization of a point-of-care biosensing platform for the detection of multiple brain-pathology biomarkers.

Morphological and Elastic Transition of Polystyrene Adsorbed Layers on Silicon Oxide.

Herein we present a study on the formation of irreversibly adsorbed layer of polystyrene molecules on silicon oxide surfaces. Various scanning probe microscopy techniques have been employed to study both the morphology and the mechanical properties of these self-assembled thin polymeric layers. More in detail, standard contact mode, force versus distance spectroscopy and ultrasonic force microscopy have been employed to obtain spatially-resolved maps and, thus, observe the physisorption of polystyrene on native silicon oxide substrate in function of time. Thick films, spin coated from a toluene solution, have been annealed at a temperature above the glass transition for increasing time intervals, and finally thoroughly rinsed in toluene. We have found that isolated islands of adsorbed chains are already present after an annealing time of half an hour. Prolonged annealing determines a progressive increase of the covered areas, whereas the formation of a complete flat layer requires 24 h. The pattern observed is in line with expected evolution of an unstable system, corresponding to the phenomenon of spinodal dewetting. Adhesion measurements show that the films present a reduced snap-off and the formation of a meniscus between tip and surface for annealing time up to 8 h. On the other hand, elastic measurements allow us to observe a progressive increase of the elastic modulus, with a complete transition for annealing time above 20 h. This is indication that a dense packing of the polystyrene molecules occurs, in line with the predictions of current models on the kinetics of irreversible adsorption. LAY DESCRIPTION: Herein we present a study on the formation of irreversibly adsorbed layer of polystyrene molecules on silicon oxide surfaces. Various scanning probe microscopy techniques have been employed to study both the morphology and the mechanical properties of these self-assembled thin polymeric layers. Thick polystyrene films, spin coated from a toluene solution, have been thermally annealed at a temperature above the glass transition for increasing time intervals, and finally thoroughly rinsed in toluene. We have found that isolated islands of adsorbed chains are already present after an annealing time of half an hour. Prolonged annealing determines a progressive increase of the covered areas, whereas the formation of a complete flat layer requires twenty-four hours. The adsorption pattern observed is in line with expected evolution of an unstable system, corresponding to the phenomenon of spinodal dewetting. Adhesion and elastic measurements have allowed us to observe a progressive increase of the packing density of the polystyrene molecules, in agreement with the predictions of current models on the kinetics of irreversible adsorption.

Compositional analysis of endogenous porphyrins from Helicobacter pylori.

Bacteria able to accumulate porphyrins can be inactivated by visible light irradiation thanks to the photosensitizing properties of this class of aromatic pigments (photodynamic therapy, PDT). Since the bacterial resistance to antibiotic is growing, PDT is becoming a valid alternative. In this context, the pathogen Helicobacter pylori (Hp) is a suitable target for PDT since it spontaneously produces and accumulates porphyrins. It is then important to understand the spectroscopic behavior of these endogenous species to exploit them as photosensitizers, thus improving the results given by the application of PDT in the treatment of Hp infections. In this work we extracted porphyrins from both a laboratory-adapted and a virulent strain of Hp, and we performed spectroscopic and chromatographic experiments to collect information about the composition and the spectrophotometric features of the extracts. The main components of the porphyrin mixtures were identified and their relative contribution to the global red fluorescence was examined.

Fluorescence lifetime microscopy reveals the biologically-related photophysical heterogeneity of oxyblepharismin in light-adapted (blue) Blepharisma japonicum cells.

The step-up photophobic response of the heterotrich ciliate Blepharisma japonicum is mediated by a hypericinic pigment, blepharismin, which is not present in any of the known six families of photoreceptors, namely rhodopsins, phytochromes, xanthopsins, cryptochromes, phototropins, and BLUF proteins. Upon irradiation, native cells become light-adapted (blue) by converting blepharismin into the photochemically stable oxyblepharismin (OxyBP). So far, OxyBP has been investigated mainly from a photophysical point of view in vitro, either alone or complexed with proteins. In this work, we exploit the vivid fluorescence of OxyBP to characterize its lifetime emission in blue B. Japonicum cells, on account of the recognized role of the fluorescence lifetime to provide physicochemical insights into the fluorophore environment at the nanoscale. In a biological context, OxyBP modifies its emission lifetime as compared to isotropic media. The phasor approach to fluorescence lifetime microscopy in confocal mode highlights that fluorescence originates from two excited states, whose relative balance changes throughout the cell body. Additionally, Cilia and kinetids, i.e., the organelles involved in photomovement, display lifetime asymmetry between the anterior and posterior part of the cell. From these data, some hypotheses on the phototransduction mechanism are proposed.

Nanoimaging: photophysical and pharmaceutical characterization of poly-lactide-co-glycolide nanoparticles engineered with quantum dots.

Quantum dots (QDs) and polymeric nanoparticles (NPs) are considered good binomials for the development of multifunctional nanomedicines for multimodal imaging. Fluorescent imaging of QDs can monitor the behavior of QD-labeled NPs in both cells and animals with high temporal and spatial resolutions. The comprehension of polymer interaction with the metallic QD surface must be considered to achieve a complete chemicophysical characterization of these systems and to describe the QD optical properties to be used for their unequivocal identification in the tissue. In this study, by comparing two different synthetic procedures to obtain polymeric nanoparticles labeled with QDs, we investigated whether their optical properties may change according to the formulation methods, as a consequence of the different polymeric environments. Atomic force microscopy, transmission electron microscopy, confocal and fluorescence lifetime imaging microscopy characterization demonstrated that NPs modified with QDs after the formulation process (post-NPs-QDs) conserved the photophysical features of the QD probe. In contrast, by using a polymer modified with QDs to formulate NPs (pre-NPs-QDs), a significant quenching of QD fluorescence and a blueshift in its emission spectra were observed. Our results suggest that the packaging of QDs into the polymeric matrix causes a modification of the QD optical properties: these effects must be characterized in depth and carefully considered when developing nanosystems for imaging and biological applications.

Role of extracellular calcium and mitochondrial oxygen species in psychosine-induced oligodendrocyte cell death.

Globoid cell leukodystrophy (GLD) is a metabolic disease caused by mutations in the galactocerebrosidase (GALC) gene. GALC is a lysosomal enzyme whose function is to degrade galacto-lipids, including galactosyl-ceramide and galactosyl-sphingosine (psychosine, PSY). GALC loss of function causes progressive intracellular accumulation of PSY. It is widely held that PSY is the main trigger for the degeneration of myelinating cells and progressive white-matter loss. However, still little is known about the molecular mechanisms by which PSY imparts toxicity. Here, we address the role of calcium dynamics during PSY-induced cell death. Using the human oligodendrocyte cell line MO3.13, we report that cell death by PSY is accompanied by robust cytosolic and mitochondrial calcium (Ca(2+)) elevations, and by mitochondrial reactive oxygen species (ROS) production. Importantly, we demonstrate that the reduction of extracellular calcium content by the chelating agent ethylenediaminetetraacetic acid can decrease intra-mitochondrial ROS production and enhance cell viability. Antioxidant administration also reduces mitochondrial ROS production and cell loss, but this treatment does not synergize with Ca(2+) chelation. Our results disclose novel intracellular pathways involved in PSY-induced death that may be exploited for therapeutic purposes to delay GLD onset and/or slow down its progression.