Prof. Franco Cacialli

Conjugated molecular and supramolecular materials now provide a class of semiconductors in its own right, with potential application to devices such as light-emitting diodes, LEDs, displays, transistors, and solar cells, now mature for both large-scale industrial take-up and commercial exploitation. After a brief introduction to conjugated polymers, I will present an electro-optical technique for the non-invasive probing of internal built-in fields in sandwich devices (LEDs or solar cells) and, in combination with data from ultraviolet photoelectron spectroscopy (UPS) and Kelvin Probe characterisation, for the analysis of the energy level line-up at organic semiconductors/electrodes interfaces. I will also present an approach to high-resolution lateral patterning of an electroluminescent conjugated polymer, based on near-field lithography with apertured probes. The technique is based on the spatially selective inhibition of the solubility of the polymer precursor by exposure to the UV field present at the apex of double-tapered, gold-coated probes [aperture diameters between 40 and 80 nm (±5 nm)]. After development in methanol and thermal conversion under vacuum we obtain features with a minimum dimension of 50 nm. I will also report results on heating of the SNOM apertured probes resulting from the combined effect of significant absorption in the metallic coating and small optical throughput of the probes. Implications for microspectroscopy and lithography will also be discussed. Insulated molecular wires made with conjugated-polymers-based polyrotaxanes offer an example of an alternative, bottom-up approach to electroluminescent nanostructures. An attractive feature here is that this class of materials is engineered at a supramolecular level by threading a conjugated macromolecule, such as poly(para-phenylene), poly(4,4-diphenylene vinylene) or poly(9,9-fluorene) through a- or b-cyclodextrin rings, so as to reduce intermolecular interactions and solid-state packing effects, that red-shift and partially quench the luminescence. Such a supramolecular approach preserves the fundamental semiconducting properties of the conjugated wires, and is effective at both increasing the photoluminescence efficiency and blue-shifting the emission of the conjugated cores, in the solid state, while still  charge-transport. We used the polymers to prepare a range of LEDs and light-emitting electrochemical cells (LECs). The reduced tendency for polymer chains to aggregate shows in both solid-state films, as well as in solution (as clearly demonstrated by time-resolved fluorescence studies) and allows solution-processing of individual polyrotaxane wires onto substrates, as revealed by scanning-force microscopy.

Dr. Lionel Vayssieres

The demand of novel functional materials has become the major challenge scientists face to answer crucial contemporary issues such as alternative energy sources, novel sensors for a safer and cleaner environment and for health (e.g. early detection of cancer and regenerative therapies). For instance, one of the promising alternatives for the transition of energy resource from its fossil fuel-based beginning to a clean and renewable technology relies on the widespread implementation of solar-related energy systems, however the high cost of energy production and low-energy of currently used material combinations pose an intrinsic limitation. In this context, revolutionary materials development is required to achieve the necessary dramatic increases in power generation and conversion efficiency. The need of low cost purpose-built, functional materials with optimized geometry, orientation, and aspect ratio combined with inexpensive large scale manufacturing methods will play a decisive role in the success of solar related energy source. However, fabricating and manufacturing large area of such functional materials is a daunting challenge. Novel smarter and cheaper fabrication techniques and, just as important, better fundamental knowledge and comprehensive understanding of materials and their syntheses as well as their properties using nanoscale phenomena such as quantum confinements to create multi-functional structures and devices is the key to success. R&D exploiting Nanoscience and Nanotechnology has the greatest potential to reach such challenging goals.
Such ideas will be demonstrated by the low-cost design and fabrication of 3-D crystalline arrays of metal oxide quantum dots and rods based structures and devices with controlled orientations, size and shape onto various substrates designed at nano-, meso-, and micro-scale by aqueous low-temperature chemical growth. In addition, in-depth characterization of their electronic structure at synchrotron radiation facilities and their application for solar hydrogen generation, photovoltaics, magnetic and gas sensor devices will be presented.

Prof. Patricia Targon Campana

The understanding of the relation between the protein structure and its function is an important challenge in Science, since proteins are involved in all crucial process for life appearing and maintenance. In this sense, the comprehension of stability and conformational mechanisms that can interfere in the protein function is essential to achieve the knowledge and subsequent treatment of several pathologies. Among the most interesting proteins for these studies one can find the lectins: proteins that present the ability to bind carbohydrates specifically and in a reversible way. As carbohydrates can be found in cell membranes, this special feature makes these proteins proper to mediate molecular recognition processes as cell proliferation, cell-cell and cell-virus interactions. Although those proteins have been extensively studied during the last decades, new lectins with interesting properties have been isolated and even the most studied ones presented open questions about their biological function and conformational changes as well. In this sense, this talk aims to present some conformational studies of lectins and other proteins with biotechnological application that have been performed in several structure levels. The changes in secondary structure by means of Far-UV Circular Dichroism, steady-state and phase domain fluorescence at aromatic vicinities and tertiary and quaternary levels using Small-Angle X-Ray Scattering (SAXS) will be presented.

Professor in Chemistry and Biochemistry,

Faculty of Medicine, University of Rome Tor Vergata, Rome, Italy.

Via Montpellier 1, 00133 Rome

Office: Department of Experimental Medicine and Biochemical Sciences

Tel: +39 06 72596354

Research Activity

1) Laboratory development and managing.
2) Molecular biology techniques
3) Microbiology techniques.
4) Immunobiosensors for analisys of enviromental pollution and food contaminants.
5) Cellular immunology
6) Immunochemistry
7) Cell culture and monoclonal antibody production
8) Drug design and HTS
9) Activity evaluation of new drugs against parasites.

Recent Publications

[1]. Ciaccio C, Coletta A, De Sanctis G, Marini S., Coletta M. (2008) Cooperativity and allostery in haemoglobin function. IUBMB Life. Feb;60(2):112-23

[2]. Gemma S, Campiani G, Butini S, Kukreja G, Coccone SS, Joshi BP, Persico M, Nacci V, Fiorini I, Novellino E, Fattorusso E, Taglialatela-Scafati O, Savini L, Taramelli D, Basilico N, Parapini S, Morace G, Yardley V, Croft S, Coletta M, Marini S, Fattorusso C. (2008) Clotrimazole scaffold as an innovative pharmacophore towards potent antimalarial agents: design, synthesis, and biological and structure-activity relationship studies. J Med Chem. 2008 Mar 13;51(5):1278-94. Epub 2008 Feb 16.

[3]. Monaco S, Gioia M, Rodriguez J, Fasciglione GF, Di Pierro D, Lupidi G, Krippahl L, Marini S, Coletta M. (2007) Modulation of the proteolytic activity of matrix metalloproteinase-2 (gelatinase A) on fibrinogen. Biochem J. Mar 15;402(3):503-13.

[4]. Saladino R, Fiani C, Crestini C, Argyropoulos DS, Marini S, Coletta M. (2007) An efficient and stereoselective dearylation of asarinin and sesamin tetrahydrofurofuran lignans to acuminatolide by methyltrioxorhenium/H(2)O(2) and UHP systems. J Nat Prod. Jan;70(1):39-42.

[5]. Gioia M, Monaco S, Fasciglione GF, Coletti A, Modesti A, Marini S, Coletta M. (2007) Characterization of the Mechanisms by which Gelatinase A, Neutrophil Collagenase, and Membrane-Type Metalloproteinase MMP-14 Recognize Collagen I and Enzymatically Process the Two alpha-Chains. J Mol Biol. May 11;368(4):1101-1113. Epub 2007 Mar 2.

Full Professor in Biochemistry at the School of Veterinary Medicine,

University of Teramo, Italy.

Visiting Professor at the School of Medicine,

University of Rome “Tor Vergata”, Italy.

Tel.: 0861266875

Tel.: 0672596388

Research Activity

Published 207 full papers including 32 review articles (total I.F. ≈ 800), and 43 invited papers in international refereed journals, besides 41 mini-papers and 128 Congress communications on the following main topics:
1. Structure and function of enzymes; 2. Membranes and lipid messengers in programmed cell death (apoptosis) and in neurodegenerative processes; 3. Endocannabinoids; 4. Ultraweak luminescence in biomedicine; 5. Gravitational effects on enzyme reactions.

Recent Publications

[1] Dainese E, Oddi S, Maccarrone M.
Lipid-mediated dimerization of beta(2)-adrenergic receptor reveals important clues for cannabinoid receptors.
Cell Mol Life Sci. 2008 May 26.

[2] Battista N, Pasquariello N, Di Tommaso M, Maccarrone M.
Interplay between endocannabinoids, steroids and cytokines in the control of human reproduction.
J Neuroendocrinol. 2008 May;20 Suppl 1:82-9.

[3] Di Marzo V, Maccarrone M.
FAAH and anandamide: is 2-AG really the odd one out?
Trends Pharmacol Sci. 2008 May;29(5):229-233.

[4] Cupini LM, Costa C, Sarchielli P, Bari M, Battista N, Eusebi P, Calabresi P, Maccarrone M.
Degradation of endocannabinoids in chronic migraine and medication overuse headache.
Neurobiol Dis. 2008 May;30(2):186-9.

[5] Battista N, Rapino C, Di Tommaso M, Bari M, Pasquariello N, Maccarrone M.
Regulation of male fertility by the endocannabinoid system.
Mol Cell Endocrinol. 2008 Apr 16;286(1-2 Suppl 1):S17-23.

[6] Kurtuncu M, Battista N, Uz T, D’Agostino A, Dimitrijevic N, Pasquariello N, Manev R, Maccarrone M, Manev H.
Effects of cocaine in 5-lipoxygenase-deficient mice.
J Neural Transm. 2008;115(3):389-95.

Opening ceremony of Joint Laboratory on June 30th, 12.00 a.m. Building PP1, via della Ricerca Scientifica., 00133 Roma.

programma

Professor of Biochemistry at the Faculty of Medicine

University of Rome Tor Vergata

Via Montpellier 1, 00133 Rome

Office: Department of Experimental Medicine and Biochemical Sciences

Tel: +39 06 72596478

Research Activity

Scientific topics: A. Proteases and inhibitors: mast cell proteases, structural and functional studies, interaction with endogenous inhibitors, search for new inhibitors;. B. Protein folding/unfolding of cytochrome c as probed by limited proteolysis, its interaction with lipids and search for the structural requirements for its pro-apoptotic activity.

Recent Publications

[1] Sinibaldi F, Fiorucci L, Patriarca A, Lauceri R, Ferri T, Coletta M, Santucci R. Insights into Cytochrome c-Cardiolipin Interaction. Role Played by Ionic Strength (2008) Biochemistry. [Epub ahead of print]

[2] Santucci R., Sinibaldi F., Fiorucci L. Protein Folding, Unfolding and Misfolding Role Played by Intermediate States Mini-Reviews in Medicinal Chemistry, 2008, 8, 000-000 1

[3]Agueci F., Polticelli F., Sinibaldi F., Piro M.C., Santucci R., Fiorucci L. (2007) Probing the effect of mutations on cytochrome c stability Protein and Peptide Letters, 14, 335-339 ISSN 0929-8665

[4]Sinibaldi F., Mei G., Ponticelli F., Piro M.C., Howes, B.D., Smulevich G., Santucci R., Ascoli F., Fiorucci L. (2005) ATP specifically driver refolding of non native conformations of cytochrome c Protein Science 14: 1049-1058 ISSN 0961-8368

Title of the Research project

Description of work

Qualification and experience

Title of the Research project

Description of work

Qualification and experience

Read More paper.pdf

Villa Mondragone, Università degli Studi di Roma Tor Vergata

Monteporzio Catone, Rome, Italy

7 - 10 July 2008

NANO SEA 2008 will primarily focus on the topics that are of strong current interest in the fields of nanostructures self-assembling and nanostructured substrate

Web site:

http://www.nanosea2008.roma2.infn.it/index.html

Contacts:

Prof. Maurizio De Crescenzi

connect-research-2008.pdf

connect-research-08-program.pdf

Collaborative research among Italian and British scientists and engineers from Nast Centre for Nanoscience Nanotechnology and Innovative Instrumentation, CNR (I), ISIS Spallation neutron Source (UK), Universities of Central Lancashire, Padova and Milano Bicocca, and from the avionics and aerospace industries have been using VESUVIO neutron flux and energy spectrum in benchmark activation measurements to demonstrate that it provides a neutron spectrum similar to the ambient one at sea level, but with an enhancement in intensity of a factor hundred .The VESUVIO beam line at the ISIS spallation neutron source UK Facility over the last few months has been set up for neutron irradiation tests in the neutron energy in the MeV range. The neutron production at ISIS relies upon spallation reactions induced by 800 MeV proton bunches, accelerated through a synchrotron.

Read More microchip-testing.pdf

Read the Press Release

Office: Department of Chemical Science and Technology

Tel: +39 06 72594414

Fax: +39 06 72594328

Research Activity

RP is Assistant Professor of Chemistry at the Faculty of Science of the University of Rome Tor Vergata.

RP research activity is mainly focused on the following topics:

• Nucleation and growth processes of polycrystalline and nanocrystalline diamond films deposited onto technologically important substrates by both Hot Filament Chemical Vapour Deposition (HFCVD) and Microwave Plasma Assisted CVD (MW-PACVD);

• Surface modification of cemented carbides for improved adhesion of diamond coatings;

• Synthesis, characterization and evaluation of functional properties of micro- and nano-structured advanced electroceramics for Solid Oxide Fuel Cells (SOFCs);

• Electrophoretic Deposition (EPD) of ceramic films;

• Chemical Vapour Infiltration (CVI) of porous Silicon layers with nanostructured sp²-carbon.

• RP has been the leader of several R&D projects funded by Italian SMEs concerning nucleation, growth, adhesion, wear resistance and life of CVD diamond films onto cemented carbide base tools (total funding > 350,000 €).

Active Research Collaborations

• Department of Mechanical Engineering, University of Aveiro, PORTUGAL;

• Laboratoire Capteurs Diamant (CEA-LIST-DETECS – SSTM), Commissariat à l’Energie Atomique, Saclay, FRANCE;

• Fraunhofer Institute for Surface Engineering and Thin Films (IST), Braunschweig, GERMANY.

Professional Recognitions

• 2008 - Invited Speaker, 5th International Conference on Hot-Wire Chemical Vapor Deposition (HWCVD-5), Cambridge, MA (USA).

• 2008 - Invited Speaker, 2nd International Conference on Advanced NanoMaterials (ANM 2008), Aveiro, Portugal.

• 2007 - “Materials Science and Nanotechnology for Polymeric Electrolyte Membrane Fuel Cells (PEMFCs) and Solid Oxide Fuel Cells (SOFCs)” (invited seminar), Dept. of Mechanical Engineering, University of Aveiro, Portugal.

• 2007 - Organizer and Chair of the “Nanoporous Semiconductor” Symposium, 2nd International Conference on Surfaces, Coatings and Nanostructured Materials (nanoSMat 2007), Algarve, Portugal.

• 2007 - member of the International Organizing Committee of the 2nd International Conference on Surfaces, Coatings and Nanostructured Materials (nanoSMat 2007), Algarve, Portugal.

• 2006 - Visiting Research Scientist at the Dept. of Mechanical Engineering, University of Aveiro, Portugal.

• 2006 - “Interlayers for diamond film deposition on hostile substrates” (invited seminar), Dept. of Mechanical Engineering, University of Aveiro, Portugal.

• 2006 - “Diamond CVD: nucleation and early growth stages” (invited seminar), Commissariat a l’Energie Atomique (CEA), Saclay, France

• 2005 - Invited Speaker, International Conference on Surfaces, Coatings and Nanostructured Materials (nanoSMat2005), Aveiro, Portugal.

• 2005 - member of the International Scientific Committee of the International Conference on Surfaces, Coatings and Nanostructured Materials (nanoSMat2005), Aveiro, Portugal.

• 2004 - Visiting Research Scientist at the Dept. of Mechanical Engineering, University of Aveiro, Portugal.

• 2001 - Visiting Researcher at the Dept. of Chemistry and Materials, Manchester Metropolitan University, Manchester, UK.

Scientific Publications

RP is the author of more than 70 papers published in peer-reviewed international scientific journals, 2 patents and more than 30 papers in Conference Proceedings. Click here to read the complete list of RP publications.

Other Activities

RP is a member of the Editorial Board of the International Journal of Surface Science and Engineering (IJSurfSE).

Teaching Activities

Chemistry of Materials, Faculty of Science;Processing Structure and Properties of Sintered Materials, Faculty of Science.Member of the Teaching Supervising Committee of the Ph.D. Course in Materials for Health, Environment and Energy.

Recent Publications (selected)

[1] R. Polini and M. Barletta: “On the use of CrN/Cr and CrN interlayers in hot filament chemical vapour deposition (HF-CVD) of diamond films onto WC-Co substrates”, Diamond Relat. Mater., 17 (2008) 325-335.

[2] R. Polini, A. Falsetti, E. Traversa, O. Schäf, P. Knauth, “Sol-gel synthesis, X-Ray Photoelectron Spectroscopy and electrical properties of Co-doped (La,Sr)(Ga,Mg)O3-δ perovskites”, J. Eur. Ceram. Soc., 27 (2007) 4291-4296.

[3] J. C. Arnault, L. Intiso, S. Saada, S. Delclos, P. Bergonzo, R. Polini, “Effect of 3C-SiC(100) initial surface stoichiometry on bias enhanced diamond nucleation”, Appl. Phys. Lett. 90 (2007) 044101.

[4] G. Mattei, V. Valentini, R. Polini, “Chemical Vapour Infiltration of nano-structured carbon in Porous Silicon”, Phys. Stat. Sol. (c), 4 (2007) 2049-2053.

[5] R. Polini, F. Pighetti Mantini, M. Barletta, R. Valle, F. Casadei, “Hot Filament Chemical Vapour Deposition and wear resistance of diamond films on WC-Co substrates coated by PVD-arc deposition technique”, Diamond Relat. Mater., 15 (2006) 1284-1291.

[6] G. Cabral, P. Reis, R. Polini, E. Titus, N. Ali, J.P. Davim, J. Grácio, “Cutting performance of time-modulated chemical vapour deposited diamond coated tool inserts during machining graphite”, Diamond Relat. Mater., 15 (2006) 1753-1758.

[7] R. Polini, “Chemically vapour deposited diamond coatings on cemented tungsten carbides: substrates pretreatments, adhesion and cutting performance”, Thin Solid Films, 515 (2006) 4-13.

[8] S. Licoccia, R. Polini, C. D’ Ottavi, F. Serraino Fiory, M. L. Di Vona, E. Traversa, “A simple and versatile method for the synthesis of functional nanocrystalline oxides”, Journal of Nanoscience and Nanotechnology, 5 (2005) 592-595.

[9] R. Polini, A. Falsetti, E. Traversa, “Sol-gel synthesis and characterization of Co-doped LSGM perovskites”, J. Eur. Ceram. Soc., 25 (2005) 2593-2598.

[10] J. C. Arnault, G. Schull, R. Polini, M. Mermoux, J. Faerber, “Effects of bias enhanced nucleation hot-filament chemical-vapor deposition parameters on diamond nucleation on iridium”, J. Appl. Phys., 98 (2005) 033521 1-9.

Titles of the Research projects

Post Doc Positions

Qualification and experience

Read More at the web site of British Embassy Rome

The technique is based on a small turbomolecular pumped vacuum vessel with access for the laser beam (1064, 532, 355 and 266 nm from a Q-switched Quantel Brilliant b laser), an optical port for stereomicroscopic visual inspection of the ablation sample, an electrostatic deflection system to discriminate between ablated neutrals and ions, a manually microtranslated (XYZ) mask holder, and a liquid nitrogen cooled cold finger cryostat. The available setup is reported in Figure 1.

Figure 1 - The Pulsed Laser Depostion (PLD) equipment has the possibility to micrometrically translate the sample or the shadow mask in order to achieve micropatterned depositions, and to ablate biological samples from frozen solutions. Other micro or nanopatterned deposits can be obtained by e-beam lithography (A. Gerardino, CNR-IFN).

The possibility to steer ionized atoms and molecules out of the ablated neutral beam and address ions on a surface along a trajectory at right angle with respect to the ablation direction allows accomplishment of extremely uniform growth at very low growth rates. This has produced recently very uniform and thin metal layers. Figure 2 reports a lithographically fabricated pattern of platinum squares as imaged by SEM (left) and by AFM (right)

Figure 2. SEM image on the left, AFM detail on the right

The Pt layers we obtain by PLD are extremely thin and uniform. In this AFM scan we have an atomically uniform, 9 Angstrom thick layer, while Figure 3 reports a detail of one such square showing the step height and thickness.

The associated histogram reports the height distributions of the topographic image for both the uncoated native silicon oxide on silicon and for the platinum coated part.

Figure 3
We can see that the step height is 9 Angstrom, while the distribution width of the substrate and of the deposition is identical, showing that the Pt coat does not contribute to the substrate roughness. The same apparatus is used for micropatterned metal deposition (Figure 4) and “soft” biomolecule deposition (Figure 5) using microtranslated shadow masks. Furthermore, visual microscopic control of the laser spot on the target allows quick and easy fabrication of micrometric shadow masks (Figure 6). Platinum micropatterned PLDs on Si as imaged by SEM on the left. On the right, selectively coloured luminescence from chromophore tagged antigens reacting with Pulsed Laser Depositions of their specific antibodies (confocal microscopy). Pulsed Nd-YAG laser drilled shadow mask on 100 micron thick stainless steel blade.

Figure 4 Figure 5

Figure 6

A home made piezoscanner, designed for xy scanning of the sample, is hosted on the sample stage of a modified Cambridge S200 scanning electron microscope, while the STM tip is positioned on the desired feature under study by a ρ,θ,φ nanomanipulator. This configuration is the most suitable to minimize mechanical vibration of the STM; alternatively, a particularly low weight scanner is mounted on the nanomanipulator while the sample is fixed. Laser beams can be directed to the interaction zone through an optical port at the back of the SEM chamber, which also serves for microscopic observation of the laser beam alignment on the STM junction. Directing the Laser beam onto the tip allows tip enhanced localised laser nanostructuring (ablation or multiphoton deposition) and localised spectroscopy.

A scheme of the STM-SEM facility is reported below (left). The images on the right show an optical microscope view of the laser irradiated STM tip on the sample, and an SEM scan of the STM tip operating to deposit material on the tips of a couple of lithographic nanoelectrodes fabricated by A. Gerardino at the CNR-IFN electron beam facility.

This facility is particularly useful for precise positioning of the tip on the specific features or devices to be analyzed or operated upon. It allows STM electronic spectroscopy to be effected on features previously easily located on the basis of the difference in secondary electron emission. It allows a prompt characterization of both tip and sample surface damage. It supplies a first easy characterization of surface modifications, like nanometric pit formation or inorganic material depositions, when the sample is irradiated by laser beams. Finally it gives us the possibility to effect much shorter, and as a consequence slower and less damaging, STM scans for a complete characterization of the sample.

Within the general issue of nanofabrication in our laboratory we are interested in controlling the applied forces during sharp probe interaction with the samples. We have purchased commercial piezoresistive cantilever based dynomometer probes and are planning to build more sensitive ones. Attaching this kind of piezoresistive or mechanical probes to a piezoelectric inertial nanomanipulator allows measurement of the stiffness and compliance of a wide range of materials. If the interaction is made under electron microscopy observation, we can easily gain a practical experience on the interaction of sharp tips with materials. We show here how a commercial AFM probe can be stressed and used to dig pits on a gold surface (movie), how we can measure the sensitivity of insects’ tactile hair sensors (movie), how we can measure the compliance of carbon nanotubes wool (movie), and even how we can accomplish a micrometric electrical switch based on a carbon nanotubes helical spring (movie).

This facility based at the ENEA laboratories is hosted in a 35 l ionically pumped ultra-high vacuum vessel endowed with a home made STM, a Z inertial piezo slider for sample approach and a remotely controlled ρ,θ,φ nanomanipulator for sample movement with submicron precision. The vessel has two load-lock chambers for tip and sample load and a hand actuated wobbling pincer for manual operations. It also has optical ports for access to the tip region by two different laser beams.

Above on the left is the scheme of the UHV STM system coupled to the available laser sources and to the spectroscopic analysis system devoted to “tip-enhanced” photoluminescence studies. On the right, a photograph of the UHV vacuum vessel with its lid lifted shows details of the optical microscope case and of the STM. Below, an optical microscope image of the STM tip over a layer of Rhodamine 6G fluorescent dye in white light (left) and excited by a green laser (540 nm) as recorded through a long wavelength pass filter with cutoff at 560 nm with the tip within tunnelling distance (center) and 5 μm away from the surface (right). Light from the tip-enhancement area is spatially selected by a moveable pinhole and analyzed by a monochromator producing the R6G emission spectrum reported below.

Related Information
Dr. P. Morales ENEA

…extremely high electromagnetic field intensities can be achieved. This well known fact is at the base of the so called “Surface” Enhanced Raman Spectroscopy, where the field enhancement is in fact due to nanometric corrugations of the surfaces; but it can also be exploited to bring the high intensity e.m. field where we want by a piezo controlled nanometric tip. Such intense radiation can be used for all the purposes for which we use lasers in the macro world: ablation, melting, recrystallization, welding, photoionization, spectroscopy and spectroscopic imaging. There are several advantages in this: the most obvious is that in the near field of the tip we can obtain concentrated radiation in a region smaller than the diffraction limit; more easily, we can obtain radiation effects in regions smaller than the practically used focal volumes, which are often hundreds of microns wide. As a secondary advantage, we can deliberately increase the focal region in order to obtain a smaller dependence on the alignment precision, on vibrations, on thermal effects, still retaining nanometric resolution. The other obvious advantage is the ability to subtract or add material from a surface at the nano scale, and characterize what we have done, both morphologically and spectroscopically, in real time by the same apparatus. In the following figures we report an example of holes burnt in an optical Compact Disk by mildly focussed laser radiation impinging on a STM tip, as imaged by Scanning Electron Microscopy; and different examples of spatially resolved depositions of both biological (the aminoacid tryptophan) and metallic (lead) material on flat surfaces as imaged by AFM and STM. Burns onto a blank CD surface are produced by laser pulses at 540 nm, irradiating the surface at very low angle, when the STM tip is brought close to the tunnelling distance. The laser beam is mildly focussed by a 500 mm focal length lens so that the beamwaist is of the order of 102 microns, much wider than the burns size, as evidenced by the green shaded area.

A deposition of the aminoacid tryptophan is obtained by photoionizing gas phase neutral molecules, as vaporized from a solid sample, under a micrometric electrode positioned 20 microns above the surface. The laser beam size is again of the order of 102 μm, while the deposition, as imaged by AFM here, is 20 μm.

Exploiting the narrow resonant excitation of the first excited electronic state of lead we can photoionize laser ablated atoms and pin them down on the underlying surface by an applied electric field. Real time STM imaging shows that the spot size depends on the tip-surface distance. Rastering the tip over the surface produces a square deposition of photoionized atoms

The next figure shows an optical microscopy view of the tip-enhancement of the fluorescence excitation as measured on a layer of the organic dye Rhodamine 6G.

The spectral distribution of the photoluminescence collected from the most luminous spot under the tip ensures that the dye molecules are not damaged. This technique thus ensures the ability to characterize spectroscopically micro-nano selected regions.

Professor in Structure of Matter

Department of Physics,

University of Rome La Sapienza,

Piazzale Aldo Moro 2, 00185 Rome ITALY

Tel:  +39 06 49914378

Fax: +39 06 4957697

Research Outline

Molecular Dynamics and Monte Carlo of Statistical Mechanical Systems

The focus of my research activity is on developing algorithms fro Molecular Dynamics simulation of complex systems in condensed matter phases. From the from now ancient SHAKE algorithm (a procedure to introduce holonomic constraints in MD) or the Subtraction Technique (a noise reducing approach to compute the response to weak external fields in Nonequilibrium MD) to the introduction of the Blue Moon’s ensemble (to simulate in MD rare events) and techniques to simulate Brownian motion to the most recent, and still very active, field of rigorous algorithms to compute nonadiabatic quantum-classical dynamics, the attempt is to widen the domain of computer simulation in condensed matter with a particular emphasis on MD (as distinguished from the very close but different MC- Monte Carlo). Together with that, I have been, and still am, also interested in challenging applications od atomistic MD simulations ranging from surface/interface physics problems in Materials sciences to simulations of biological molecules to find atomistic level explanations of their behavior or functioning mechanisms. More generally, I am interested in considering a variety of developments/applications in the simulation of systems of Statistical Mechanics interest.

Curriculum Vitae et Studiorum

Full List of Publications

Most Recent Publications

[135] E.Vanden Eijnden, and G. Ciccotti, “Second-order integrators for Langevin equations with
holonomic constraints”, Chem. Phys. Lett., 429, 310, (2006)

[134] M. S. Causo, G. Ciccotti, S.Bonella, and R. Vuilleumier, “An adiabatic linearized path integral approach for quantum time correlation functions II: A cumulant expansion method for improving convergence”, J. Phys. Chem. B, 110, 3638, (2006)

[135] V. Marry, and G. Ciccotti, “Trotter derived algorithms for molecular dynamics with constraints : Velocity Verlet revisited”, J. Comp. Phys., 222, 428, (2007)

[132] L. Maragliano, A. Fischer, E. Vanden Eijnden, and G. Ciccotti, “String method in collective variables: minimum free energy paths and isocommittor surfaces”, J. Chem. Phys., 125, 024106, (2006).

[131_B] R. Kapral, and G. Ciccotti, “Transport Coefficients of Quantum-Classical Systems”, in “Computer Simulations in Condensed Matter: From Materials to Chemical Biology (The Erice Lectures)”, M. Ferrario, G. Ciccotti, and K. Binder Eds, LNP, Springer Verlag, (2006).

[130_B] G. Ciccotti, D. Coker, and R. Kapral, “Quantum statistical dynamics with trajectories”, in Quantum Dynamics of Complex Molecular Systems, p. 275-294, I. Burghardt and D. Micha Eds, Springer Verlag, Berlin, (2007)

[129] G. Kalibaeva, R. Vuilleumier, S.Meloni, A. Alavi, G. Ciccotti, and R. Rosei, “Ab initio simulation of carbon clustering on Ni(111) surface: a model of the poisoning of nickel based catalysts”, J. Phys. Chem. B, 110, 3638, (2006)

[128] F. Pizzitutti, A. Giansanti, P. Ballario, P. Ornaghi, P. Torreri, G. Ciccotti, and P. Filetici, “Relevant role of loop ZA and Pro371 in the function of yeast Gcn5p bromodomain: evidences from Molecular Dynamics and experiments”, Journal of Molecular Recognition, 19, 1, (2006)

Books

B1) “Molecular Dynamics Simulation of Statistical Mechanical Systems.”, ‘E. Fermi’ 1985 Summer School. G.Ciccotti and W.G.Hoover, Eds., North Holland 1986.

B2) “Simulation of Liquids and Solids. Molecular Dynamics and MonteCarlo methods in Statistical Mechanics. A reprint book.”, G. Ciccotti, D. Frenkel and I. R. Mc Donald, Eds. North Holland, 1987.

B3) “MC and MD of condensed matter systems”, Euroconference 1995, K. Binder and G. Ciccotti, Eds., SIF 1996.

B4) “Simulation of classical and quantum dynamics in condensed phase”, Euroconference 1997, B. J. Berne, G. Ciccotti and D. F. Coker, Eds., World Scientific, 1998.

B5) “Bridging time scales: Molecular simulations for the next decade”, SIMU Conference, Konstanz 2001, P. Nielaba, M. Mareschal, and G. Ciccotti, Eds., Springer, Berlin, 2003

B6) “Computer Simulations in Condensed Matter: From Materials to Chemical Biology (The Erice Lectures)”, M. Ferrario, G. Ciccotti, and K. Binder, Eds, LNP, Springer Verlag, Berlin, (2006)

Related Information
contact: Dr. P. Morales
ENEA

The technique is described in Figure 1. To achieve the best yield in the activity of deposited biomolecules, these are pulsed laser ablated from a frozen solution target maintained at approximately 150K on the tip of a liquid nitrogen cooled cold finger cryostat in vacuo (pressure is approximately 10^-3 mbar). The tip faces the optical window of the vacuum vessel and is exposed to the laser beam pulses, mildly focussed by a 30 cm focal length lens. The solution is doped by a suitable molecule absorbing in a wavelength range away from the absorption of proteins (salicylaldeheyde, 330 nm - 360 nm) and irradiated by the third harmonic of a Q-switched Nd-YAG laser (355 nm). This maximizes sublimation preserving the proteins. The target surface is visually inspected during ablation by a stereoscopic microscope.

Figure 1 Figure 2

The ablated molecular jet is highly ionized and is thus subject to a static electric field applied between target and substrate, driving the ions to the deposition substrate. Micrometric patterning is achieved by a shadow mask fixed on the substrate and by a microtranslated shutter mask selecting the locations to be coated. On the other hand, nanometric patterning was only attempted with the enzyme horseradish peroxidase by electron beam lithography of a polimethylmethacrilate film, followed by pulsed laser deposition and by liftoff in acetone.

A multiple tip rotary cold finger allows three different molecular species to be ablated in a single vacuum run. The formed layers are found to be biologically functional; in the case of the enzyme horseradish peroxidase in particular, biological activity is assessed by measuring the absorption of the enzymatic reaction products, namely the absorption of the enzymatically oxidized substrate ABTS; the enzymatic activity of horseradish peroxidase coated specimens is found to increase strongly with the applied field as reported in Figure 2, due to better collection on the sample and to little damage of the protein structure in spite of the energetic impact. These data suggest that not only ablated neutral proteins, but also ionized molecules accelerated to the surface retain their biological activity. Above 1500 V electrical discharge easily occurs with our setup, which destroys the molecular layer, so that very little or no biological activity is detected on the sample.

The following image (Figure 3), collected by a laser induced fluorescence confocal microscope, shows micrometric patterns of green fluorophore tagged anti-human Immunoglobuline that has reacted on a laser deposited stenciled deposition of Human Immunoglobuline G.

Figure 3

Fig. 4 (left) is an AFM detail of the layer structure, showing clustering of Immunoglobulines G, that have a size of approximately 3×7 nm, into larger lumps; only few of the observable structures have sizes comparable to that of single Immunoglobuline molecules. The layer thickness, as measured on manufactured step edges is between 5 and 10 nm, corresponding approximately to 2-3 stacked molecules.

Fig. 5 shows a nanopatterned horseradish peroxidase layer as imaged by AFM in intermittent contact mode and phase contrast, demonstrating the possibility to obtain extremely dense biomolecular arrays.

Figure 6

The decrease of the enzymatic activity of a coated specimen repeatedly rinsed in a water solution supplies a qualitative measure of the adhesion of the laser deposited molecules onto the surface. Figure 6 reports such biological activity decrease for layers deposited on a glass and on a stainless steel surfaces.
Two important features should be observed. Both substrates show a dual beaviour, a fast decrease at first followed by a much slower decrease. Secondly, the metal substrate shows a slower biological activity decrease with respect to the glass substrate; the adhesion of the laser deposited layer is thus higher on metal than on glass. We believe this behaviour is due to the fact that the effective electric field between the molecular source and the glass substrate quickly decreases while deposited ions charge up the surface. Therefore the average kinetic energy of the molecules is lower when they impact on the glass substrate than on the metal one. The deformation of the proteic globule, which we believe to allow a high number of weak interactions to be established, is consequently higher on metals, giving rise to better adhesion. AFM measurement of the layer thickness with increasing rinsing time shows that the thickness decreases very quickly at first, soon reaching a value of approximately 5 nm, very close to the monolayer value. The rinse time necessary to reach the monolayer always corresponds to the time needed to reach the slope change in the above plot. The monolayer is then much harder to remove from the surface, and the enzymatic activity of the specimen consequently is well detectable after 24 hours.
To show that this methodology can indeed supply heterogeneous biosensing arrays of micrometrically resolved thin film islands of different active proteins, we fabricated a simple immunosensing microarrays using two different immunoglobulines (IgG human and IgG mouse); of each we deposited three spots at random in a 3×3 matrix, while the remaining three locations were coated by a platinum thin film for control of the mask alignment. The platinum spots are visible by an ordinary microscope in light field while they are non fluorescent under excitation in the blue and green region. The specific antibodies for the IgG Human and IgG Mouse molecules were tagged with green and red emitting fluorophores respectively and incubated on the laser deposited specimen for 25 min. Fig. 7a shows a microscope image of the specimen in light field, where only the platinum spots are visible, while 7b shows the same specimen after incubation with a solution containing both antibodies and dual excitation at 488 and 514 nm. We find the green and red spots as expected on the IgG Human and IgG Mouse spots respectively, while no emission is visible from the platinum coated locations.

Figure 7

Sezione FIM MATNANO
Bldg. F65, Centro Ricerche della Casaccia, ENEA
Via Anguillarese km 1.3
00123 S.Maria di Galeria, Roma
Tel: +39 06 30486350
Lab: +39 06 30486082

Research Activity

From his first interest in molecular spectroscopy, which he pursued at the University La Sapienza in Roma, at the University of Venice and at the University of Kent UK, PM moved to spectroscopic applications in the field of uranium isotope separation, on which he worked up to the end of the italian laser isotope separation project led by ENEA. More recently he developed an interest in micro and nanobioscience and in nanotechnologies. From 1997 to 2000 he was the european coordinator of the LASMEDS project, devoted to the development of a molecular electronics technology that exploits vapour phase photoionization in the near field of STM probes. Later he coordinated and was involved in other national projects on nanotechnology, nanobioscience and organic devices. His present interests are in nanofabrication following both the inorganic, machine assisted, and the biological selfassembling approaches.

Recent Publications

[1] S. Gagliardi, B. Rapone, L. Mosiello, D. Luciani, A. Gerardino, P. Morales, Laser-Assisted Fabrication of Biomolecular Sensing Microarrays, IEEE Transactions on Nanobioscience, 6 (3): 242 – 248 (2007)

[2] A. Dell’Aquila, P. Mastrorilli, C.F. Nobile, G. Romanazzi, G.P. Suranna, L. Torsi, M.C. Tanese, D. Acierno, E. Amendola, P. Morales, Synthesis and field-effect properties of alpha,omega-disubstituted sexithiophenes bearing polar groups, Journal of Materials Chemistry, 16 (12): 1183-1191 (2006)

[3] S. Gagliardi, S. Nufris B. Rapone, P. Morales, G. D’Agostaro, Laser techniques for the fabrication of nanobiodevices, Proceedings of SPIE - The International Society for Optical Engineering, 5850, pp. 271-279 (2004)

[4] T. Di Luccio, G. Scalia, L. Tapfer, P. Morales, M. Traversa, P. Prete, N. Lovergine, Microstructural and morphological properties of homoepitaxial (001) ZnTe layers investigated by x-ray diffuse scattering,
Journal of Applied Physics, 97 (8): Art. No. 083540 (Apr 15 2005).

[5] I. Dierking, G. Scalia, P. Morales, Liquid crystal-carbon nanotube dispersions, Journal of Applied Physics, 97 (4): Art. No. 044309 (Feb 15 2005).

[6] M. Traversa, N. Lovergine, P. Prete, G. Scalia, M. Pentimalli, L. Tapfer, P. Morales, A.M. Mancini, Effects of substrate treatment and growth conditions on structure, morphology, and luminescence of homoepitaxial ZnTe deposited by metalorganic vapor phase epitaxy, Journal of Applied Physics, 96 (2): 1230-1237 (Jul 15 2004).

[7] P. Morales, Laser assisted SPM nanofabrication, in Encyclopaedia of Nanoscience and Nanotechnology, American Scientific Publishers, (Jan. 2004).

[8] I. Dierking, G. Scalia, P. Morales, D. LeClere, Aligning and reorienting carbon nanotubes by nematic liquid crystals, Advanced Materials, 16 (11) 865-869 (Jun 4 2004).

[9] A. Gerardino, A. Notargiacomo, P. Morales, Laser assisted deposition of nanopatterned biomolecular layers, Microelectronic Engineering, 67-8: 923-929 (Jun 2003).

Related Information
contact: Prof. E. Traversa
Tissue-Specific Stem Cells

… skin and cornea [3], while the reconstruction of more complicated soft tissues is far more challenging. In particular, cardiovascular diseases are the main cause of death in the western world and there is an impelling need for new solutions to treat hearts damaged by myocardial infarction and to address the shortage of heart donors. Tissue engineering approaches are mainly based on the use of 3D biocompatible, bioerodable scaffolds and cardiac cells to reconstitute contractile cardiac muscle-like tissues in vitro that might be utilized for the replacement of diseased myocardium in vivo [4,5]. One order of problems consists in the identification of the proper source of cells. Recent advances in stem cell research now offer a promising cell source for the treatment of heart failure, due to their ability to self-renew and their potential to differentiate into the specialized cells [6]. However, this is not sufficient for the success of the tissue reconstruction. A suitable interfacing between cells and scaffold is necessary for maintaining the activity of functional cells, regulating cell behavior, and reconstructing three-dimensional (3D) tissues. The scaffold must exhibit biocompatibility and biodegradability, porosity with adequate pore size to favor cell attachment and growth, as well as to facilitate the diffusion of nutrients to and waste products from the implant.

Amongst the various materials employed for tissue engineering scaffolds, synthetic polymers such as polycaprolactone (PC), polyglicolide (PGA), polylactide (PLA), and their copolymers have drawn considerable attention due to their processability. The selection and design of the material is crucial to yield physical and geometrical characteristics at different length scales, necessary to reproduce the tissue complexity at the cell-scaffold interface. Figure 1 shows the features of a cardiac tissue at different length scales: (i) an optical micrograph of a heart tissue section showing that vascular ducts have size at the millimetre scale, (ii) a scanning electron (SEM) micrograph of cardiac stem cells showing that their size is of about 20 μm [7], and (iii) a scheme of the extracellular matrix (ECM) components at the nanometre scale.

Figure 1: Different length scales of cardiac tissue to be integrated into the scaffold: mm scale for vascularization and nutrition, μm scale for cell accomodation, nm scale for the expression of ECM components.

The scaffold should incorporate in a single construct the information that can trigger the reproduction of the cardiac tissue with all the different features. Therefore, approaches in scaffold design must be able to create hierarchical porosities in a single construct, at the millimetre scale to help nutrition and vascularization, at the micrometer scale to accommodate cells, and at the nanometre scale to favour the expression of extra-cellular matrix components, with the desired chemical and mechanical functions. The importance of producing three-dimensional structures with porosities at scales from the nanometre to millimetre level has been reported mainly for osteoblasts, to influence the interaction between cells ad scaffold in terms of mass-transport requirements for cell nutrition, migration and attachment [8]. In some cases the emphasis has been put onto the nanoscale features needed by the scaffold, again in most of the cases for bone treatments and drug delivery [9]. Although there are certainly similarities amongst the various cells, there are also specificities for the different tissues, and what is valid for bones should be critically evaluated, say, for heart.
Several processing techniques have been developed over the past two decades to cast a large variety of materials into scaffolds suitable for the many different applications of tissue engineering and cell types (e.g. soft vs. hard tissues). Note worthily, none of them has emerged universally yet as the superior choice over the others, being all three currently active research topics. The strategy followed by researchers of NAST Centre is to develop several low-cost and user-friendly technologies that can be opportunely tuned to fabricate matrices with controlled architecture and morphological features, to match the requirements of hierarchical porosity and roughness that mimic the cell native surroundings. Some of these technologies, examined in here, are porogen leaching, phase separation and electrospinning [10].
1) Porogen leaching techniques involves the use of suitable porogens to be introduced into the polymer matrix and subsequently removed to generate porosity within the scaffold itself. The size and shape of the pores are entirely determined and tailored by those of the porogen chosen. For example, paraffin micro spheres of different size can be ad hoc synthesized and selected as porogen agents(Figure 2 – left). The resulting polymeric scaffold is shown by the SEM micrograph in Figure 2 (right), demonstrating spherical porosity in a close packed arrangement. The inset picture contains a cross section of the scaffold confirming a spherical open structure with communication paths between neighboring spherical holes. The opportune selection and preparation of the porogen as well as the balance of the polymer/porogen ratio allows manufacturing controlled scaffolds in terms of porosity and pore shape and size.

Figure 2: SEM micrographs of poly-L-Lactic acid, PLA, scaffolds prepared by paraffin leaching techniques. The paraffin spheres (left) leached out from a polymeric matrix leaving controlled open porosity (right) (the insert reports the cross-sectional view).

2) The phase separation technique is based on a thermodynamic demixing of a homogeneous polymer-solvent solution into a polymer-rich phase and a polymer-poor phase. In other words, the procedure discussed here consisted in a solid-liquid extraction of the solvent from a polymer-solvent frozen solution, the solvent acting as a porogen agent. It is possible to tune the architecture (in terms of morphology and porosity) of the scaffold obtained by this technique by tuning the temperature of the extraction process. Figures 3a and 3b show the comparison between the morphology of polymeric scaffolds obtained by the solid-liquid extraction performed in isothermal (T1=-18°C) and non-isothermal (-18°C<T2<25°C) conditions, respectively. Both samples have a regular, homogeneous, and highly porous surface, with pore size being different depending on the preparation conditions. The structures obtained at T1 (Figure 3a) showed a smaller pore size (about 20 μm in diameter) than the sample prepared in non-isothermal conditions (Figure 3b). The insets of Figure 3 show zoomed areas of the scaffold surface. In both cases, pores at the nanometre scale can be observed in the polymeric foam struts. It is evident from this example that it is possible to obtained multiscale architectures of polymer scaffold, with hierarchical porosity that matches the features of native tissue.

Figure 3: SEM images of a poly-L-Lactic acid, PLA, scaffold prepared by the phase separation technique at two different temperature (T) of solvent extraction, (a) T=-18°C and (b) -18°C<T<25°C. The insets show high magnification SEM micrographs of a PLA scaffold prepared by the phase separation technique at two different temperature (T) of solvent extraction, (a) T1=-18°C and (b) -18°C<T2<25°C.

3) Electrospinning is a polymer processing technique that has been revitalized with the advent of tissue engineering. The working principle of the technique consists of applying a high-voltage electric field (of the order of 1÷10 kV) between a metallic capillary containing a desired polymeric solution/melt and a collecting electrode (usually a conductive plate) electrically grounded. When the electrical field overcomes the resistance of the surface tension of the polymeric solution, the electrically charged solution is ejected from the capillary towards the counter electrode. The polymeric jet stretches under the action of the electrostatic force and produces long and thin fibers [11]. In practice, the polymer solution is electrospun from a flat-ended needle of a syringe, whose shaft is actuated by a programmable syringe pump that supplies the solution to the needle and fuels the process. In most of the cases reported in the literature, electrospinning is used to prepare mats of fibers to be used as scaffolds for tissue engineering, with limited control in porosity and therefore in cell adhesion and growth. None the less electrospinning offers the possibility to tune most geometrical and morphological properties on the entire hierarchy of scales of the scaffolds. Thus, besides controlling the fibre diameter, it is possible for example to modify the macro porosity by changing the confluence, the number of layers, or the alignment of the fibres in the network. Micro and nanoporosity may also exist and be controlled. Figure 4 shows electrospun PLLA nanofibers (300÷400 nm in diameter) with characteristic nanopores, whose size can be controlled via process parameters (i.e., applied voltage, electrode distance and configuration, concentration and viscosity of the solution, solution flow rate, etc).

Figure 4. SEM image of poly-L-Lactic acid, PLA, electrospun nanofibers with nanopores.

Electrospinning allows also the control of fibre alignment for the purpose of tuning the macro texturing of the scaffold. In general, if a simple collecting plate (e.g. an aluminium foil) is used, the electrospun fibres deposit randomly, forming a spaghetti-like stack. We devised patterned electrodes to control the achieve fibre alignment. Figure 5 (left) shows the experimental setup used, where two Cu parallel bars were first used as grounded collecting electrodes. Each bar was 50 mm long, with a square cross section 5 mm wide. They were placed 50 mm apart at a distance of about 170 mm from the syringe needle charged at about 20 kV. An auxiliary 180 mm diameter Cu ring was also connected to the generator to stabilize the electric field and control the jet motion. In this configuration, PCL fibers with a diameter of 2÷4 μm electrospan orderly between the Cu bars, rendering the highly oriented bundle (not shown). This result, relevant per se, served as a building block to device scaffold with more complicated patterns. Another experiment was performed with the same experimental set-up but the addition of another pair of Cu bar electrodes placed at 90° from the former, as shown in Figure 5 (bottom-left). The aim was the fabrication of a scaffold made with two groups of fibres oriented along orthogonal directions. Figure 5 (right) shows, indeed that the resulting scaffold exhibited a macroscopic texture with both a short-range ordered plot and periodic features orthogonal to each other. This approach offers a convenient technique to modulate the in-plane elasticity of the scaffold which has been found to be relevant for differentiation of cardiac progenitor cells [12].

Figure 5: Schematics of the electrospinning experimental set up (left), where the grounded collecting electrodes were (a) two Cu parallel bars, (b) two pair of Cu bar electrodes placed orthogonal to each others. SEM micrograph of macroscopically textured PCL scaffold from 90° crossing of fiber bundles.

The biological validation of the produced scaffolds was performed in vitro using a murine Lineage negative, Sca-1 positive, mesenchymal stem cell line of bone marrow origin (mMSC) [12]. Cells were seeded on sterilized and equilibrated scaffolds at the concentration of 3X104/cm2. Cell viability and proliferation were tested after 24, 48, and 72 hours. Data showed no significant evidence of cell death with respect to cells grown on chamber slides in standard culture conditions (used as controls). To detect the proliferation ability, cycling cells were calculated as the ratio between the number of mitotic nuclei and total number of fixed and stained nuclei by fluorescence microscope analysis. Data showed that mitotic nuclei number of cells grown on scaffolds increased of 1.5 fold, after 24h and 3 folds, after 48h, similarly to controls. After 72h, no significant number of mitotic nuclei was observed, being cells grown at confluence. The validation was successful for all the scaffolds discussed inhere. For illustrative purpose, Figure 6 shows the immunofluorescence (IF) images of the mMSC seeded on scaffolds shown in Figures 3b. Stem cells were able to colonize the pores created and to adhere functionally to the scaffolds. Indeed adherent cells, decorated with tetra-rhodamine conjugated phalloydine, directed against F-actin, showed a typical elongated shape and well organized stress fibres. The results demonstrated not only the cytocompatibility of the materials used, but also significant adhesion and proliferation of seeded cells onto the constructs.

Figure 6: Immunofluorescence analysis of F-actin expression (red) in mMSC grown after 72h, on PLA scaffold from Figure 3b. Nuclei were stained with 4’, 6’-diamidino-2-phenylindole (DAPI). Images of cell samples were taken with a Leica DMRB microscope using a digital camera. The images were representative of at least five random fields for each sample.

Conclusions

The body of the experimental results demonstrated that 3D multiscale polymeric scaffolds for tissue engineering can be pursued with several approaches, each one having a characteristic signature. Porogen leaching (in a lesser extent), phase separation, and electrospinning techniques, all allowed the fabrication of porous scaffolds exhibiting features on different length scales, whose properties can be finely tuned by controlling the process parameters. Changing the size and shape of a target porogen, as well as modifying the temperature profile of a phase separation process, is a low-cost and user-friendly operation, enabling scaffold architecture in a broad range of length scales. Similarly, changing the materials (e.g., polymer, solvent, the composition of the solution) and/or the experimental setup (e.g., applied voltage, electrode distance, electrode pattering) can radically transform the appearance of the fibre network prepared using electrospinning. By means of purposely designed collecting electrodes, it is possible to switch from a totally random macrotexture to an ordered arrangement. Overall, the results demonstrate the applicability of tuneable polymeric scaffolds with hierarchical porosities in cardiac tissue engineering. Adult stem cells adhered and proliferated onto the fabricated constructs, providing the biological validation.

Co-authors of the study

E. Traversa, S. Licoccia, Centro NAST & DSTC, Università di Roma Tor Vergata
B. Mecheri, C. Mandoli, S. Soliman, A.Rinaldi, DSTC Università di Roma Tor Vergata
P. di Nardo, M. Minieri, Centro NAST & Dept. Int. Medicine, Università di Roma Tor Vergata
G. Forte, F. Carotenuto, F. Pagliari, S. Pagliari, Dept. Int. Medicine, Università Roma Tor Vergata

References

[1] R. Langer and J.P. Vacanti. Tissue engineering. Science, 260, 920 (1993).
[2] Y. Cao; J. P. Vacanti; K. T. Paige; J. Upton; C. A. Vacanti. Transplantation of chondrocytes utilising a polymer-cell construct to produce tissue engineered cartilage in the shape of a human ear. Plast Reconstruct Surg, 100, 297 (1997).
[3] K. Nishida, M. Yamato, Y. Hayashida, K. Watanabe, K. Yamamoto, E. Adachi, S. Nagai, A.Kikuchi, N. Maeda, H. Watanabe, T. Okano, and Y. Tano. Corneal Reconstruction with Tissue-Engineered Cell Sheets Composed of Autologous Oral Mucosal Epithelium. N. Eng J. Med. 351,12 (2004).
[4] W.H. Zimmermann, C. Fink, D. Kralisch, U. Remmers, J. Weil, T. Eschenhagen. Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes. Biotechnol. Bioeng., 68, 106 (2000).
[5] Y. Miyahara, N. Nagaya, M. Kataoka, B. Yanagawa1, K. Tanaka, H. Hao, K. Ishino, H. Ishida, T. Shimizu, K. Kangawa, S. Sano, T. Okano, S. Kitamura and H. Mori. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nature Medicine, 12, 459 (2006).
[6] N.N. Malouf, W.B. Coleman, J.W. Grisham, R.A. Lininger, V.J. Madden, M. Sproul, P.A. Anderson. Adult-derived stem cells from the liver become myocytes in the heart in vivo. Am. J. Pathol., 158, 1929 (2001).
[7] R. Fiaccavento, F. Carotenuto, M. Minieri, C. Fantini, G. Forte, A. Carbone, L. Carosella, R. Bei, L. Masuelli, C. Palumbo, A. Modesti, M. Prat, P. Di Nardo. Stem cell activation sustains hereditary hypertrophy in hamster cardiomyopathy. J. Pathol., 205, 397 (2005)
[8] M.M. Stevens and J.H. George. Exploring and Engineering the Cell Surface Interface. Science, 310, 1135 (2005).
[9] E. Palin, H. Liu, and T.J. Webster. Mimicking the nanofeatures of bone increases bone-forming cell adhesion and proliferation. Nanotechnology, 16, 1828 (2005).
[10] E. Traversa, B. Mecheri, C. Mandoli, S. Soliman, A. Rinaldi, S. Licoccia, G. Forte, F. Pagliari, S. Pagliari, F. Carotenuto, M. Minieri, P. Di Nardo. Tuning Hierarchical Architecture of 3D Polymeric Scaffolds for Cardiac Tissue Engineering. Journal of Experimental Nanoscience, in press 2007
[11] L. Dan, X. Younan. Electrospinning of nanofibers: reinverting the wheel?. Adv. Mater., 16, 1151 (2004).
[12] G.Forte, F. Carotenuto, F. Pagliari, S. Pagliari, P. Cossa, R. Fiaccavento, A. Ahluwalia, G. Vozzi, B. Vinci, A. Serafino, A. Rinaldi, E. Traversa, L. Carosella, M. Minieri, P. Di Nardo. Polymeric scaffold guidance to stem cell differentiation and tissue warping. Nature Materials, (submitted September 2007).

Related Information
NUME, CARISMA, CNR-TAE,
Università Mediterranea Reggio Calabria,
RCAST Tokyo, Università di Roma Tor Vergata

Related Information
contact: Prof. S. Licoccia

The material most widely used forelectrolyte membranes is Nafion, a perfluorinated sulfonated polymer (Figure 1). Because of its water assisted conduction mechanism, Nafion can be used only at temperatures below 100 °C [1]. For practical applications, however, operating PEMFCs at higher temperature is desired, both for hydrogen and methanol-fuelled cells. When hydrogen is used as a fuel, an increase in the cell temperature above 100°C produces enhanced CO tolerance, faster reaction kinetics, easier water management, and reduced heat exchanger requirement. The use of methanol as a fuel for vehicles has several practical benefits such as easy transport and storage, but the slow oxidation kinetics of methanol and its crossover through the membrane reduce the efficiency of direct methanol fuel cells (DMFCs). Therefore, increasing the operation temperature of proton conducting membranes is a key issue in the development of PEMFC technology [2,3].

The strategy followed by researchers of NAST Centre is to develop organic/inorganic hybrid membranes in order to achieve the goal of obtaining PEMs capable to operate at temperatures up to and above 120°C . Organic/inorganic hybrids are investigated for a variety of applications, ranging from optics to electronics, to sensors and many others, since they offer the possibility to combine the properties of the two components in a unique material [4]. Thus, we have studied nanocomposites where inorganic materials were added to a polymeric matrix, either of Nafion or of totally aromatic sulfonated polymers. In the case of polymeric electrolytes, the addition of nanocrystalline oxides is effective in improving ionic conductivity, mechanical strength, and thermal stability [5]. Since performance is strongly influenced by the filler particle size, we developed a simple and versatile route for the preparation of ceramic oxides with nanometric grain size [6]. MxOy (M = Ti, Nb, In and Zr) were synthesized by rapid hydrolysis of an alcoholic solution of metal alkoxides. The preparative conditions were optimized to enhance the reaction kinetics leading to fast nucleation and favoring the formation of nanoparticles. Figure 2 shows the TEM micrograph of TiO2 (chosen as a representative example) obtained by thermal treatment of the sol-gel precursor at 500°C. The particle size was in agreement with the XRD measurements (12 nm), although the presence of aggregates was determined. The in-house prepared nanocrystalline titania was used as a filler in the preparation of Nafion based composite membranes to be used as electrolytes in PEMFCs, with the aim of increasing the operation temperature. The electrochemical performance of such membranes was investigated both in methanol and hydrogen fuelled fuel cells.

Figure 2. TEM micrograph of sol-gel derived Titania calcined at 500°C.

All the Membrane Electrode Assemblies (MEAs) prepared with the membranes of different composition were capable to operate in a DMFC up to 145°C, a temperature much higher than that reached by a bare Nafion recast membrane.
Figure 3 shows, again as a representative example, the polarization (a) and power density (b) curves obtained for the DMFC prepared with such MEAs with a 2 M methanol solution feed at the anode, and using oxygen as oxidant. A maximum power density of 350 mW cm^-2 was reached at a current density of about 1.1 A cm^-2 with the membrane containing 5 wt% TiO2. Moreover, the cell was subjected to a one month cycled operation (6 hours per day including start up and shut down procedures) without significant performance decrease. The presence of the fillers contributed to stabilize the polymer morphology at high temperature and inhibited the direct permeation of reaction gases by modifying the transport pathways [7].
Operation at temperatures above 100°C is needed also for hydrogen-fuelled polymer electrolyte fuel cells, both for direct hydrogen (DH-PEFC) and processed hydrogen (PH-PEFC) cells, to enhance reaction kinetics, reduce heat-exchange requirements and, in PH-PEFC, increase CO tolerance. We have thus studied the electrochemical performance of a Nafion based composite membrane, containing 3 wt% nanometric TiO2 calcined at 400°C [8].
Table I summarizes the water uptake and ion exchange capacity (IEC) of the nanocomposite membrane compared with the values relative to a pure Nafion recast membrane prepared in the same experimental conditions and a commercial Nafion 115 membrane. The water uptake and ion exchange capacity of the composite membrane were significantly higher than those determined for the reference pure Nafion recast membrane, while commercial Nafion 115 gave intermediate values

Table I
Water uptake and ion exchange capacity data for different Nafion-based membranes.

The proton conductivity of the membranes was examined in the temperature range from 80°C to 130°C. The conductivity was always higher for the composite membrane, reaching a value of 0.18 S/cm at 130°C. The commercial Nafion 115 and the composite membrane were tested in DH-PEFC, in a single cell, in humidified H2/air, between 80°C and 130 °C. In the whole temperature range tested, the best performance was obtained with the composite membrane. Figure 4 shows the polarization and power

density curves obtained for the two membranes at 100°C and those relative to the nanocomposite membrane at 130°C. A power density of 0.514 mW cm^-2 was recorded for the composite membrane versus 0.354 mW cm^-2 obtained with Nafion 115 at 0.56 V and 110 °C. Most important is to notice that while the pure Nafion membrane was damaged at temperatures above 100°C, the composite membrane continued to operate up to 130°C reaching a power density of 0.254 mW cm^-2 at 0.5 V. Tests were also carried out in a PH-PEFC. The cell was fed with steam reforming synthetic fuel (SR: 10 ppm CO; 20% CO2; 75% H2; 1% CH4). Figure 5 shows a comparison between polarization curves at 110°C in pure hydrogen and SR for the nanocomposite membrane. For both fuels, the measured OCV values were good, almost reaching 1 V. A power density value of about 182 mW cm^-2 at 0.6 V in synthetic fuel, versus the 366 mW cm^-2 value measured in pure hydrogen, was obtained. Although the performance for tested MEA fed with SR is lower by about 50% if compared to the performance in pure hydrogen, it is reasonable to assume that the use of suitable electrodes containing Pt-Ru as an electro-catalyst would minimize such loss. Nevertheless, it appeared evident that the filler exerts a beneficial effect in allowing operation at T > 100 °C even in SR fuel.

Arylene main chain polymers have receive a great deal of attention as possible alternative to perfluorinated systems mainly because of their lower cost and their ease of functionalization [9,10]. Among the different polymers, we focused our attention on sulfonated polyetheretherketone (SPEEK, Figure 6).

Figure 6 The structure of sulfonated Poly Ether Ether Ketone (SPEEK)

Even if at high degree of sulfonation (DS), its conductivity is large enough to meet the requirements needed for application in PEMFCs, its mechanical properties tend to progressively deteriorate with sulfonation because of the absence of significant hydrophilic-hydrophobic separation that results in very narrow and poorly connected water channels and large separation between the sulfonic acid groups [10,11]. To attain the correct balance between the hydrophilic and hydrophobic components we prepared nancomposite membranes containing hydrated tin oxide, a filler that is proton conducting in its own right and to decrease the polymer DS.

The procedure used to synthesize SnO2 x 1.5H₂O allowed to obtain nanosized particles. Figure 7 shows the typical SEM micrograph of the as prepared SnO2 x n(H₂O) powder. As typical morphology for hydrated compounds, the powder was made of soft agglomerates. Numerous SEM observations allowed to measure the unit particles in the nanometric size range (5-15 nm). Such a small particle size is expected to ease the formation of homogeneous composite membranes.

Figure 7. SEM micrograph of the as-prepared SnO₂ x nH₂O powder.

Figure 8 shows the Arrhenius plots of the conductivity of the composite membranes and of the unfilled SPEEK reference membrane. In the region between 25°C and 75°C, the proton conductivity (σ) values of the composite membrane were always larger than the values measured for the unfilled SPEEK membrane. In particular, at 25°C, σ of the composite membrane was one order of magnitude larger than the σ of the unfilled SPEEK (composite: 0.016 S cm^-1; unfilled SPEEK 0.0017 S cm^-1). This difference decreased with increasing the temperature and eventually was null at 75°C. The λ values (the number of water molecules per sulfonic acid group of SPEEK) was calculated on the basis of water uptake measurements and considering an additive behavior of the components.

Figure 8. Arrhenius plots of the unfilled SPEEK and SPEEK/SnO2 composite membranes at 100% RH.

In agreement with electrochemical data, λ of the composite membrane was larger than the λ value of the SPEEK membrane below 60°C, temperature at which the λ values of the two samples became the same.
The overall reduced swelling of the composite membrane can be explained assuming that the filler induces morphological modification of the membrane and that the oxide hydration water molecules generate a connection between the polymer sulfonic acid moieties, creating different hydrophilic paths that favor proton transfer [12]. The effect becomes negligible at higher temperature when a decrease in the interactions between polymer chains is known to favor a greater hydration of the polymer [13]. The reduced swelling and higher morphological stability of the composite resulted also in improved membrane performance in terms of durability.
The electrochemical performance of the membranes was tested in a home-made prototype DMFC single cell (Figure 9), acquiring polarization curves.
Figure 9. The H₂ and CH3OH fuelled Fuel Cell built at Tor Vergata.

Figure 10 shows the polarization and power curves for the SPEEK/SnO2 composite membrane in a DMFC test at 100 °C, compared with a recast Nafion membrane. As already mentioned, Nafion is the most widely used electrolyte for DMFCs, even though it exhibits high methanol permeation rate as well as a drop in proton conductivity at temperatures larger than 80°C. The I-V curves clearly showed the improved performance of the composite membrane with respect to the Nafion membrane. The open circuit voltage (OCV) for the composite membrane (0.70 V) was larger than that of unfilled SPEEK (0.65 V) and reference Nafion (0.59 V). The polarization curve of the unfilled SPEEK membrane is not shown due to its lower stability in terms of proton conductivity as a function of time. This finding shows that the use of a SPEEK membrane allowed to reduce the methanol crossover with respect to Nafion. Moreover, the presence of tin oxide allowed a further reduction of the methanol crossover.
In the whole voltage range investigated, the current values of the SPEEK/SnO₂ membrane were always larger than the values obtained with the Nafion membrane. In particular, the composite membrane reached a maximum current density value of 350 mAcm^-2 (with respect to 200 mAcm^-2 for unfilled Nafion). The maximum power density value reached at 100°C with the composite membrane was 80 mWcm^-2 at a current density of about 300 m A cm^-2, whereas the maximum power density value of the reference unfilled Nafion membrane at the same temperature was 20 mWcm^-2, at a current density of about 120 mAcm^-2. The improvement of the polarization curve of the SPEEK/SnO₂ composite membrane reflected the drop in conductivity of the unfilled Nafion membrane above 90°C and the enhanced stability of the composite membrane at 100°C.

These results converge to indicate that filling SPEEK membranes with hydrated tin oxide allowed to improve the membrane stability, as well as to decrease its methanol permeability. These features led to an enhancement of cell performance of the composite membrane with respect to our reference Nafion recast membrane, pointing out that SPEEK/SnO₂ membrane is a promising electrolyte for DMFCs.

Conclusions

The use of organic/inorganic hybrid membranes has been demonstrated to be effective in achieving good electrochemical performance of PEMFCs at temperatures above 120°C, fed either with processed hydrogen, steam-reformed hydrogen, and methanol.
This research represents some of the results of collaborative efforts of different Italian and foreign Institutions under the framework of MiUR-FISR, MAE-Joint Laboratory and EU-Coordinative Action Projects.

Co-authors of the study

S. Licoccia, E. Traversa Centro NAST & DSTC, Università di Roma Tor Vergata
A. D’Epifanio, C. D’Ottavi B. Mecheri, R. Polini DSTC Università di Roma Tor Vergata
P. Antonucci Univ. Mediterranea of Reggio Calabria
V. Antonucci, A. S. Aricò, E. Passalacqua V. Baglio, A. Carbone, A. Di Blasi, A. Saccà, R. Ornelas CNR-TAE Messina

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