Pietro Parisse

Efficient water oxidation at carbon nanotube–polyoxometalate electrocatalytic interfaces

Water is the renewable, bulk chemical that nature uses to enable carbohydrate production from carbon dioxide. The dream goal of energy research is to transpose this incredibly efficient process and make an artificial device whereby the catalytic splitting of water is finalized to give a continuous production of oxygen and hydrogen. Success in this task would guarantee the generation of hydrogen as a carbon-free fuel to satisfy our energy demands at no environmental cost. Here we show that very efficient and stable nanostructured, oxygen-evolving anodes are obtained by the assembly of an oxygen-evolving polyoxometalate cluster (a totally inorganic ruthenium catalyst) with a conducting bed of multiwalled carbon nanotubes. Our bioinspired electrode addresses the one major challenge of artificial photosynthesis, namely efficient water oxidation, which brings us closer to being able to power the planet with carbon-free fuels.

Glioma-Associated Stem Cells: A Novel Class of Tumor-Supporting Cells Able to Predict Prognosis of Human Low-Grade Gliomas

Background: Translational medicine aims at transferring advances in basic science research into new approaches for diagnosis and treatment of diseases. Low‐grade gliomas (LGG) have a heterogeneous clinical behavior that can be only partially predicted employing current state‐of‐the‐art markers, hindering the decision‐making process. To deepen our comprehension on tumor heterogeneity, we dissected the mechanism of interaction between tumor cells and relevant components of the neoplastic environment, isolating, from LGG and high‐grade gliomas (HGG), proliferating stem cell lines from both the glioma stroma and, where possible, the neoplasm. Methods and Findings: We isolated glioma‐associated stem cells (GASC) from LGG (n=40) and HGG (n=73). GASC showed stem cell features, anchorage‐independent growth, and supported the malignant properties of both A172 cells and human glioma‐stem cells, mainly through the release of exosomes. Finally, starting from GASC obtained from HGG (n=13) and LGG (n=12) we defined a score, based on the expression of 9 GASC surface markers, whose prognostic value was assayed on 40 subsequent LGG‐patients. At the multivariate Cox analysis, the GASC‐based score was the only independent predictor of overall survival and malignant progression free‐survival. Conclusions: The microenvironment of both LGG and HGG hosts non‐tumorigenic multipotent stem cells that can increase in vitro the biological aggressiveness of glioma‐initiating cells through the release of exosomes. The clinical importance of this finding is supported by the strong prognostic value associated with the characteristics of GASC. This patient‐based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma. Stem Cells 2014;32:1239–1253

Two-dimensional enzyme diffusion in laterally confined DNA monolayers

Addressing the effects of confinement and crowding on biomolecular function may provide insight into molecular mechanisms within living organisms, and may promote the development of novel biotechnology tools. Here, using molecular manipulation methods, we investigate restriction enzyme reactions with double-stranded (ds)DNA oligomers confined in relatively large (and flat) brushy matrices of monolayer patches of controlled, variable density. We show that enzymes from the contacting solution cannot access the dsDNAs from the top-matrix interface, and instead enter at the matrix sides to diffuse two-dimensionally in the gap between top- and bottom-matrix interfaces. This is achieved by limiting lateral access with a barrier made of high-density molecules that arrest enzyme diffusion. We put forward, as a possible explanation, a simple and general model that relates these data to the steric hindrance in the matrix, and we briefly discuss the implications and applications of this strikingly new phenomenon.

Structural and energetic basis for hybridization limits in high-density DNA monolayers

High-density monolayers (HDMs) of single-strand (ss) DNA are important nanoscale platforms for the fabrication of sensors and for mechanistic studies of enzymes on surfaces. Such systems can be used, for example, to monitor gene expression, and for the construction of more complex nanodevices via selective hybridization with the complementary oligos dissolved in solution. In this framework, controlling HDM hybridization is essential to control the final properties. Different studies demonstrate that at the typical density of ≈10(13) molecules per cm(2) no more than ≈30-40% of the HDM ssDNA is successfully hybridized. Until now, however, the origin of the HDM hybridization limit has remained unclear. In this work, molecular dynamics (MD) simulations of HDM systems with variable hybridization reveal that, independently of other experimental parameters, the effective hybridization for a HDM of this density is intrinsically limited by molecular and electrostatic crowding. A detailed structural analysis of the HDM model shows good agreement with our atomic force microscopy (AFM) experiments, and provides further insight into the steric hindrance behaviour and time-resolved surface topography of these nanostructured systems. The explicit relationship proposed between structural crowding and limited HDM hybridization offers a rationale to control the final properties of HDM-based nanodevices.

A DNA-based nano-immunoassay for the label-free detection of glial fibrillary acidic protein in multicell lysates

UNLABELLED We have developed a quantitative approach to eventually enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells. Through DNA-directed immobilization of DNA-protein conjugates we immobilized antibodies specific for a certain protein of interest, on a complementary DNA nanoarray fabricated by means of nanografting, a nanolithography technique based on atomic force microscopy (AFM). The proof of concept was realized for glial fibrillary acidic protein (GFAP), a biomarker crucial in cells differentiation of astrocytes, and functional to grade classification of gliomas, the most common of primary malignant brain tumors. The efficiency of the nano-immuno sensing was tested by obtaining the immobilization of purified recombinant GFAP protein at different concentration in a standard solution then in a cellular lysate. A comparison of sensitivity between our technique and conventional ELISA assays is provided at the end of the paper. FROM THE CLINICAL EDITOR This team developed a quantitative approach to enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells, demonstrating the utility of this DNA-based nano-immunoassay in the detection of GFAP.

Surface passivation improves the synthesis of highly stable and specific DNA-functionalized gold nanoparticles with variable DNA density.

We report a novel and multifaceted approach for the quick synthesis of highly stable single-stranded DNA (ssDNA) functionalized gold nanoparticles (AuNPs). The method is based on the combined effect of surface passivation by (1-mercaptoundec-11-yl)hexa(ethylene glycol) and low pH conditions, does not require any salt pretreatment or high excess of ssDNA, and can be generalized for oligonucleotides of any length or base sequence. The synthesized ssDNA-coated AuNPs conjugates are stable at salt concentrations as high as 3.0 M, and also functional and specific toward DNA-DNA hybridization, as shown from UV-vis spectrophotometry, scanning electron microscopy, gel electrophoresis, fluorescence, and small angle X-ray scattering based analyses. The method is highly flexible and shows an additional advantage of creating ssDNA-AuNP conjugates with a predefined number of ssDNA strands per particle. Its simplicity and tenability make it widely applicable to diverse biosensing applications involving ssDNA functionalized AuNPs.

Experimental setups for FEL-based four-wave mixing experiments at FERMI

The recent advent of free-electron laser (FEL) sources is driving the scientific community to extend table-top laser research to shorter wavelengths adding elemental selectivity and chemical state specificity. Both a compact setup (mini-TIMER) and a separate instrument (EIS-TIMER) dedicated to four-wave-mixing (FWM) experiments has been designed and constructed, to be operated as a branch of the Elastic and Inelastic Scattering beamline: EIS. The FWM experiments that are planned at EIS-TIMER are based on the transient grating approach, where two crossed FEL pulses create a controlled modulation of the sample excitations while a third time-delayed pulse is used to monitor the dynamics of the excited state. This manuscript describes such experimental facilities, showing the preliminary results of the commissioning of the EIS-TIMER beamline, and discusses original experimental strategies being developed to study the dynamics of matter at the fs-nm time-length scales. In the near future such experimental tools will allow more sophisticated FEL-based FWM applications, that also include the use of multiple and multi-color FEL pulses.

Spectroscopic ellipsometry meets AFM nanolithography: about hydration of bio-inert oligo(ethylene glycol)-terminated self assembled monolayers on gold

For the first time, to our knowledge, spectroscopic ellipsometry (SE) has been combined with state-of-the-art AFM differential height measurements conducted after shaving nano-lithography of ultrathin, soft-matter films for thickness determination. We investigated self-assembled monolayers of SH-(CH2)11-EGn-OH molecules on gold, where EG is ethylene glycol units and n = 3 and 6, a prototypical non-fouling system. We performed SE measurements (245-1200 nm) focusing on the changes induced by the formation of the film (difference spectra). SE measurements, analysed by simple models, confirm the formation of the S-Au interface, transparency of the SAMs and provide a sharp picture of the ability of the EG functionality to protect the surface from unspecific adsorption of proteins. A quantitative assessment of the film thickness by SE was carried out ex situ, thanks to the optical contrast between the film and the ambient, and by AFM in liquid. The cross-check between SE and AFM height measurements combined with the comparison between in-liquid and ex situ SE measurements allowed obtaining non-perturbative information about the vertical density profile of the SAM. The in-liquid SE measurements indicate a refractive index matching between the aqueous medium and the outer part of the SAM, consistent with a disordered configuration of OEG and/or the penetration of water amid the OEG strands. A critical discussion provides a detailed insight into the subtle issues and pitfalls related to the thickness determination of soft-matter films to the monolayer limit.

In Vitro Enzyme Comparative Kinetics: Unwinding of Surface-Bound DNA Nanostructures by RecQ and RecQ1.

Many cellular processes entail the separation of nucleic acid strands. Helicases are involved in the separation of the double-stranded DNA, a process fueled by ATP hydrolysis. We investigated the reaction mechanism of two homologous helicases, the bacterial RecQ and the human RECQ1, in vitro, that is, within confined DNA monolayers. We generate arrays of engineered DNA sequences by atomic force microscopy (AFM) nanografting and monitor the enzyme activity on the surface by means of differential, highly precise AFM measurements of the DNA height variation. The latter is associated with the unwinding action of the enzyme onto the surface-bound DNAs because it arises from the different mechanical properties of single- versus double-stranded DNA that are sensibly detected by AFM. Our results highlight different kinetic behaviors for these enzymes under the same experimental conditions.

DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors

Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.