Stefania Rapino

Co-axial heterostructures integrating palladium/titanium dioxide with carbon nanotubes for efficient electrocatalytic hydrogen evolution

Considering the depletion of fossil-fuel reserves and their negative environmental impact, new energy schemes must point towards alternative ecological processes. Efficient hydrogen evolution from water is one promising route towards a renewable energy economy and sustainable development. Here we show a tridimensional electrocatalytic interface, featuring a hierarchical, co-axial arrangement of a palladium/titanium dioxide layer on functionalized multi-walled carbon nanotubes. The resulting morphology leads to a merging of the conductive nanocarbon core with the active inorganic phase. A mechanistic synergy is envisioned by a cascade of catalytic events promoting water dissociation, hydride formation and hydrogen evolution. The nanohybrid exhibits a performance exceeding that of state-of-the-art electrocatalysts (turnover frequency of 15000 H2 per hour at 50u2009mV overpotential). The Tafel slope of ∼130u2009mV per decade points to a rate-determining step comprised of water dissociation and formation of hydride. Comparative activities of the isolated components or their physical mixtures demonstrate that the good performance evolves from the synergistic hierarchical structure.

A novel neuroferritinopathy mouse model (FTL 498InsTC) shows progressive brain iron dysregulation, morphological signs of early neurodegeneration and motor coordination deficits.

Neuroferritinopathy is a rare genetic disease with a dominant autosomal transmission caused by mutations of the ferritin light chain gene (FTL). It belongs to Neurodegeneration with Brain Iron Accumulation, a group of disorders where iron dysregulation is tightly associated with neurodegeneration. We studied the 498–499InsTC mutation which causes the substitution of the last 9 amino acids and an elongation of extra 16 amino acids at the C-terminus of L-ferritin peptide. An analysis with cyclic voltammetry on the purified protein showed that this structural modification severely reduces the ability of the protein to store iron. In order to analyze the impact of the mutation in vivo, we generated mouse models for the some pathogenic human FTL gene in FVB and C57BL/6J strains. Transgenic mice in the FVB background showed high accumulation of the mutated ferritin in brain where it correlated with increased iron deposition with age, as scored by magnetic resonance imaging. Notably, the accumulation of iron–ferritin bodies was accompanied by signs of oxidative damage. In the C57BL/6 background, both the expression of the mutant ferritin and the iron levels were lower than in the FVB strain. Nevertheless, also these mice showed oxidative alterations in the brain. Furthermore, post-natal hippocampal neurons obtained from these mice experienced a marked increased cell death in response to chronic iron overload and/or acute oxidative stress, in comparison to wild-type neurons. Ultrastructural analyses revealed an accumulation of lipofuscin granules associated with iron deposits, particularly enriched in the cerebellum and striatum of our transgenic mice. Finally, experimental subjects were tested throughout development and aging at 2-, 8- and 18-months for behavioral phenotype. Rotarod test revealed a progressive impaired motor coordination building up with age, FTL mutant old mice showing a shorter latency to fall from the apparatus, according to higher accumulation of iron aggregates in the striatum. Our data show that our 498–499InsTC mouse models recapitulate early pathological and clinical traits of the human neuroferritinopathy, thus providing a valuable model for the study of the disease. Finally, we propose a mechanistic model of lipofuscine formation that can account for the etiopathogenesis of human neuroferritinopathy.

Highly sensitive electrochemiluminescence detection of a prostate cancer biomarker

Prostate-specific membrane antigen (PSMA), a glycoprotein expressed in the prostatic epithelium endowed with enzymatic activity, is a very promising diagnostic marker for the early detection of prostate cancer. In this study, we report a novel electrochemiluminescence ELISA-like immunosensor based on carbon nanotubes and a highly specific sandwich immunoassay for the PSMA detection. To fabricate the device, an optically transparent electrode was modified with doubly functionalized multi-walled carbon nanotubes carrying amine groups and a monoclonal anti-PSMA antibody. Subsequently, to complete the sandwich immunosensing device, a second specific monoclonal anti-PSMA antibody was labelled with a electrochemiluminescent probe. Under optimized experimental conditions, the proposed sensing device exhibits a performance exceeding that of the state of-the-art in terms of the limit of detection (LOD) and limit of quantification (LOQ) as good as 0.88 ng mL-1 and 2.60 ng mL-1, respectively, in real complex samples such as cell lysates. In addition, the unique role of carbon nanotubes is also discussed by comparison with an analogue sensor assembled without the nanocarbon-based material.

Single Cell Electrochemiluminescence Imaging: From the Proof-of-Concept to Disposable Device-Based Analysis

We report here the development of coreactant-based electrogenerated chemiluminescence (ECL) as a surface-confined microscopy to image single cells and their membrane proteins. Labeling the entire cell membrane allows one to demonstrate that, by contrast with fluorescence, ECL emission is only detected from fluorophores located in the immediate vicinity of the electrode surface (i.e., 1-2 μm). Then, to present the potential diagnostic applications of our approach, we selected carbon nanotubes (CNT)-based inkjet-printed disposable electrodes for the direct ECL imaging of a labeled plasma receptor overexpressed on tumor cells. The ECL fluorophore was linked to an antibody and enabled to localize the ECL generation on the cancer cell membrane in close proximity to the electrode surface. Such a result is intrinsically associated with the unique ECL mechanism and is rationalized by considering the limited lifetimes of the electrogenerated coreactant radicals. The electrochemical stimulus used for luminescence generation does not suffer from background signals, such as the typical autofluorescence of biological samples. The presented surface-confined ECL microscopy should find promising applications in ultrasensitive single cell imaging assays.

Glucose and Lactate Miniaturized Biosensors for SECM-Based High-Spatial Resolution Analysis: A Comparative Study

With the aim of developing miniaturized enzymatic biosensors suitable for in vitro diagnostic applications, such as monitoring of metabolites at single cell level, glucose and lactate biosensors were fabricated by immobilizing enzymes (glucose oxidase and lactate oxidase, respectively) on 10 μm Pt ultramicroelectrodes. These electrodes are meant to be employed as probes for scanning electrochemical microscopy (SECM), which is a unique technique for high-spatial-resolution electrochemical-based analysis. The use of enzymatic moieties improves sensitivity, time scale response, and information content of the microprobes; however, protein immobilization is a key step in the biosensor preparation that greatly affects the overall performance. A crucial aspect is the miniaturization of the sensing, preserving their sensitivity. In this work, we investigated the most common enzyme immobilization techniques. Several fabrication routes are reported and the main figures of merit, such as sensitivity, detection limit, response time, reproducibility, spatial resolution, biosensor efficiency, permeability, selectivity, and the ability to block electro-active interfering species, are investigated and compared. With the intent of using the microprobes for in vitro functional imaging of single living cells, we carefully evaluate the spatial resolution achieved by our modified electrodes on 2D SECM imaging. Metabolic activity of single MCF10A cells were obtained by monitoring the glucose concentrations in close proximity of single living cell, using the UME-based biosensor probes prepared. A voltage-switch approach was implemented to disentangle the topographical contribution of the cells enabling quantitative measurements of cellular uptakes.

Antimicrobial properties of graphene-like nanoparticles: coating effect on Staphylococcus aureus

The exploitation of nanomaterials with antimicrobial properties has attracted an ever-growing interest in the recent years. Carbon-based materials, such as graphene and graphene family materials (GFMs), have gained most of the attention for application in many biomedical fields. Here, we describe the antimicrobial activity of graphene-like (GL) layers derived from the chemical demolition of carbon black, against the planktonic growth of Staphylococcus aureus cells, primary cause of hospital and community-acquired infections, often leading to bacteremia and sepsis. The inhibitory capabilities of GL layers on the formation of S. aureus biofilm are also assessed. The antimicrobial properties seem based mainly on the interaction between GL layers and bacteria surfaces. FESEM and AFM analyses suggest that the GL layers coat the cells as soon as they get in contact with them, as also indicated by the wettability of the GLs.

C60@lysozyme: a new photosensitizing agent for photodynamic therapy

C60@lysozyme showed significant visible light-induced singlet oxygen generation in water, indicating the potential of this hybrid as an agent for photodynamic therapy. The reactive oxygen species (ROS) concentration generated by C60@lysozyme during irradiation depends on the light source, the irradiation time and the concentration of the hybrid. C60@lysozyme significantly reduced the HeLa cell viability in response to visible light irradiation. The generation of H2O2, due to the photoactivity of C60@lysozyme, causes cell death via easy permeation of hydrogen peroxide through the cell membrane and activation of endogenous ROS production.

Enhancing radiosensitivity of melanoma cells through very high dose rate pulses released by a plasma focus device

Radiation therapy is a useful and standard tumor treatment strategy. Despite recent advances in delivery of ionizing radiation, survival rates for some cancer patients are still low because of recurrence and radioresistance. This is why many novel approaches have been explored to improve radiotherapy outcome. Some strategies are focused on enhancement of accuracy in ionizing radiation delivery and on the generation of greater radiation beams, for example with a higher dose rate. In the present study we proposed an in vitro research of the biological effects of very high dose rate beam on SK-Mel28 and A375, two radioresistant human melanoma cell lines. The beam was delivered by a pulsed plasma device, a “Mather type” Plasma Focus for medical applications. We hypothesized that this pulsed X-rays generator is significantly more effective to impair melanoma cells survival compared to conventional X-ray tube. Very high dose rate treatments were able to reduce clonogenic efficiency of SK-Mel28 and A375 more than the X-ray tube and to induce a greater, less easy-to-repair DNA double-strand breaks. Very little is known about biological consequences of such dose rate. Our characterization is preliminary but is the first step toward future clinical considerations.

Local desorption of thiols by scanning electrochemical microscopy: patterning and tuning the reactivity of self-assembled monolayers

Self-assembled monolayers (SAMs) are widely used in the field of nanotechnologies and (bio)sensors. The monolayer surface properties are tailored by employing several techniques. A large set of SAM post-modification routes are commonly performed to adapt them to a variety of nano-technological and bio-technological studies as well as to several bio-sensoristic applications. Here, we report a procedure to locally modify SAMs by electrochemical desorption of alkanethiols in order to create microsized spots of bare gold area without affecting the surrounding monolayer stability. The tip of the scanning electrochemical microscope (SECM) was employed to draw microstructured pattern according to a defined geometry. The time stability of the pattern was also tested. Furthermore, the patterned surface was post-functionalized using the same alkanethiol or a ferrocene-terminated thiol, in order to tune the surface reactivity of the microstructure. The local surface properties, including reactivity and electron transfer kinetics toward redox mediator reduction, were characterized by SECM.

Complex Media and Enzymatic Kinetics.

Enzymatic reactions in complex environments often take place with concentrations of enzyme comparable to that of substrate molecules. Two such cases occur when an enzyme is used to detect low concentrations of substrate/analyte or inside a living cell. Such concentrations do not agree with standard in vitro conditions, aimed at satisfying one of the founding hypotheses of the Michaelis-Menten reaction scheme, MM. It would be desirable to generalize the classical approach and show its applicability to complex systems. A permeable micrometrically structured hydrogel matrix was fabricated by protein cross-linking. Glucose oxidase enzyme (GOx) was embedded in the matrix and used as a prototypical system. The concentration of H2O2 was monitored in time and fitted by an accurate solution of the enzymatic kinetic scheme, which is expressed in terms of simple functions. The approach can also find applications in digital microfluidics and in systems biology where the kinetics response in the linear regimes often employed must be replaced.