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Alessandro Paolini,1 Giulia Battafarano,1 Valentina D’Oria,1 Francesco Mura,2 Simona Sennato,3 Valentina Mussi,4 Roberta Risoluti,5 Stefano Materazzi,5 Andrea Del Fattore,1 Andrea Masotti1 1Bambino Gesù Children’s Hospital, IRCCS, Research Laboratories, Rome 00146, Italy; 2Center for Nanotechnology for Engineering (CNIS), Sapienza University of Rome, Rome 00185, Italy; 3CNR-ISC UOS Sapienza and Physics Department, Sapienza University of Rome, Rome 00185, Italy; 4National Research Council, Institute for Microelectronics and Microsystems IMM-CNR, Roma 00133, Italy; 5Department of Chemistry, Sapienza University of Rome, Rome 00185, ItalyCorrespondence: Andrea MasottiBambino Gesù Children’s Hospital, IRCCS, Research Laboratories, Gene Expression-Microarrays Laboratory, Viale di San Paolo 15, Rome 00146, ItalyTel +39-06-68592650Fax +39-0668592904Email andrea.masotti@opbg.netBackground: The advent of 3D printing technology allowed the realization of custom devices that can be used not only in the everyday life but also in the nanotechnology and biomedical fields. In nanotechnology, the use of bi-dimensional nanostructures based on carbon nanotubes, generally referred as buckypapers, have received considerable attention for their versatility and potential application in many biomedical fields. Unfortunately, buckypapers are extremely hydrophobic and cannot be used in aqueous media to culture cells.Methods: A polymeric device able to accommodate buckypapers and facilitate cell growth was fabricated by using 3D printing technology. We imparted hydrophilicity to buckypapers by coating them with polyamidoamine (PAMAM) dendrimers.Results: We found that by using novel techniques such as polymer coating the buckypaper hydrophilicity increased, whereas the use of 3D printing technology allowed us to obtain custom devices that have been used to culture cells on buckypapers for many days. We characterized in details the morphology of these structures and studied for the first time the kinetic of cell proliferation. We found that these scaffolds, if properly functionalized, are suitable materials to grow cells for long time and potentially employable in the biomedical field.Conclusion: Although these materials are cytotoxic under certain circumstances, we have found a suitable coating and specific experimental conditions that encourage using buckypapers as novel scaffolds for cell growth and for potential applications in tissue repair and regeneration.Keywords: 3D printing, buckypaper, PAMAM dendrimer, cell proliferation, tissue regeneration, transfection

Photothermal therapy (PTT) assisted by nanomaterials is a promising minimally invasive technique for cancer treatment. Here, we explore the PTT properties of a silicon- and gold-based nanostructured platform suitable for being directly integrated in fibre laser systems rather than injected into the human body, which occurs for the most commonly unreported PTT nanoagents. In particular, the photothermal properties of an array of disordered silicon nanowires coated by a thin gold film (Au/SiNWs) were tested on a monolayer of human colon adenocarcinoma cells (Caco-2) irradiated with a 785 nm laser. Au/SiNWs allowed an efficient photothermal action and simultaneous monitoring of the process evolution through the Raman signal coming from the irradiated cellular zone. Strong near infra-red (NIR) absorption, overlapping three biological windows, cell-friendly properties and effective fabrication technology make Au/SiNWs suitable both to be integrated in surgical laser tools and as an in vitro platform to develop novel PTT protocols using different cancer types and NIR sources.

We report on highly disordered array of Au coated silicon nanowires (Au/SiNWs) as surface enhanced Raman scattering (SERS) probe combined with electrochemical detection for biosensing applications. SiNWs, few microns long, were grown by plasma enhanced chemical vapor deposition on common microscope slides and covered by Au evaporated film, 150 nm thick. The capability of the resulting composite structure to act as SERS biosensor was studied via the biotin-avidin interaction: the Raman signal obtained from this structure allowed to follow each surface modification step as well as to detect efficiently avidin molecules over a broad range of concentrations from micromolar down to the nanomolar values. The metallic coverage wrapping SiNWs was exploited also to obtain a dual detection of the same bioanalyte by electrochemical impedance spectroscopy (EIS). Indeed, the SERS signal and impedance modifications induced by the biomolecule perturbations on the metalized surface of the NWs were monitored on the very same three-electrode device with the Au/SiNWs acting as both working electrode and SERS probe.

Peculiar transport phenomena appear at nanoscale, since surface effects strongly affect the behaviour of fluids. Electrostatic and steric interactions, capillary forces and entropic effects play a key role in the behaviour of fluids and biomolecules. Since these effects strongly depend on the size of the nanofluidic system, a careful characterization of the fluidic environment is necessary. Moreover, the possibility to dynamically modulate the size of nanochannels is very appealing in the field of biomolecule manipulation. Recently, we have developed a lab-on-chip made of poly(dimethylsiloxane) (PDMS). This polymeric device is based on a tuneable nanochannel able to dynamically change its dimension in order to fit the application of interest. In fact, a mechanical compression applied on the top of the elastomeric device squeezes the nanochannel, reducing the channel cross section and allowing a dynamical optimization of the nanostructures. In this paper, this squeezing process is fully characterized both numerically and experimentally. This analysis provides information on the reduction of the nanochannel dimensions induced by compression as a function of the work of adhesion and of the stiffness of the materials composing the device. Moreover, calculations demonstrate the possibility to predict the change of the nanochannel size and shape induced by the compression. The possibility to dynamically tune the channel size opens up new opportunities in biomolecular sensing or sieving and in the study of new hydrodynamics effects.

Principal component analysis is applied to analyse the Raman maps collected on carbon nanotubes at different degrees of oxidation and functionalization with dye labeling molecules. The results are used to demonstrate that the technique is extremely effective in clustering data and comparing preparation protocols, so that it enables drawing of a fast and reliable classification of the molecule propensity to interact with pristine and oxidized carbon nanotubes. The spectral findings are supported and elucidated by several experimental techniques, thermogravimetry and steady-state and time-resolved fluorescence measurements, and by computational modeling, showing that the proposed methodology could represent a powerful and routine test for the rational design of functional nanostructures.

Starting from their discovery in 1991, carbon nanotubes have attracted a great attention, thanks to their peculiar mechanical, electrical and elastic properties that could be used to realize new devices in many different fields. For nanotechnology applications it is very important to be able to control not only shape and position but also alignment and orientation of carbon nanotubes, both during the growth and after it. Here we present preliminary results obtained by depositing carbon nanotubes (CNT) solutions on ion sputtered quartz substrates. Atomic force microscopy (AFM) images allow to study both CNTs positioning on the “ripples” generated by Ar+ sputtering on the SiO2 surface and their radial deformation induced by the “rough” surface. Work is now in progress to optimize the sputtering parameters and solution treatment (purification and functionalization) in order to get single CNTs regularly arranged on a patterned surface.

In the last years, nanopore technology has been increasingly exploited for biomolecule detection and analysis. Recently, the main focus of the research has moved from the study of nucleic acids to the analysis of proteins and DNA-protein complexes. In this paper, chemically functionalized solid-state nanopore has been used to recognize Nuclear Factor-kappa B proteins (NF-κB), that are involved in several disorders and inflammation processes, so that their identification is of crucial importance for prognostic applications. In particular, we show that it is possible to electrically detect the specific interaction between p50, a protein belonging to the NF-κB family, and dsLNA probe molecules covalently attached to the surface of a FIB fabricated SiN pore. The obtained results have been compared with those related to BSA protein, which does not interact with the used probes. Finally, the potential of the device has been further tested by analyzing a whole cell extract. In this case, three principal peaks in the distribution of electrical event duration can be identified, corresponding to different interacting NF-κB complexes, so that the methodology appears to be effective also to study biological samples of considerable complexity. Ultimately, the presented data emphasize the selectivity and versatility of the functionalized nanopore device, demonstrating its applicability in bioanalytics and advanced diagnostics.

Several strategies have been developed for the control of DNA translocation in nanopores and nanochannels. However, the possibility to reduce the molecule speed is still challenging for applications in the field of single molecule analysis, such as ultra-rapid sequencing. This paper demonstrates the possibility to alter the DNA translocation process through an elastomeric nanochannel device by dynamically changing its cross section. More in detail, nanochannel deformation is induced by a macroscopic mechanical compression of the polymeric device. This nanochannel squeezing allows slowing down the DNA molecule passage inside it. This simple and low cost method is based on the exploitation of the elastomeric nature of the device, can be coupled with different sensing techniques, is applicable in many research fields, such as DNA detection and manipulation, and is promising for further development in sequencing technology.

We demonstrate the possibility of using a simple functionalization procedure, based on an initial vapour-phase silanization, to control the size and functionality of solid state nanopores. The presented results show that, by varying the silanization time, it is possible to modify the efficiency of probe molecule attachment, thus shrinking the pore to the chosen size, while introducing a specific sensing selectivity. The proposed method allows us to tune the nanopore biosensor adapting it to the specific final application, and it can be efficiently applied when the pore initial diameter does not exceed a limit dimension related to the mean free path of the silane molecules at the working pressure.

We present the development and the electrical characterization of a polymeric nanochannel device. Standard microfabrication coupled to Focused Ion Beam (FIB) nanofabrication is used to fabricate a silicon master, which can be then replicated in a polymeric material by soft lithography. Such an elastomeric nanochannel device is used to study DNA translocation events during electrophoresis experiments. Our results demonstrate that an easy and low cost fabrication technique allows creation of a low noise device for single molecule analysis.

Single molecule electrical sensing with nanopores is a rapidly developing field with potential revolutionary effects on bioanalytics and diagnostics. The recent success of this technology is in the simplicity of its working principle, which exploits the conductance modulations induced by the electrophoretic translocation of molecules through a nanometric channel. Initially proposed as fast and powerful tools for molecular stochastic sensing, nanopores find now application in a range of different domains, thanks to the possibility of finely tuning their surface properties, thus introducing artificial binding and recognition sites. Here we show the results of DNA translocation and hybridization experiments at the single molecule level by a novel class of selective biosensor devices that we call “ DNA - D ressed NA nopore” ( DNA 2 ), based on solid state nanopore with large initial dimensions, resized and activated by functionalization with DNA molecules. The presented data demonstrate the ability of the DNA 2 to selectively detect complementary target sequences, that is to distinguish between molecules depending on their affinity to the functionalization. The DNA 2 can thus constitute the basis for the design of integrable parallel devices for mutation DNA analysis, diagnostics and bioanalytic investigations.

The possible use of nanopores for single DNA molecules biosensing has been demonstrated, but much remains to do in order to develop advanced engineered devices with enhanced stability, and controlled geometry and surface properties. Here we present morphological and electrical characterization of solid state silicon nitride nanopores fabricated by focused ion beam direct milling and chemically functionalized by probe oligonucleotides, with the final aim of developing a versatile tool for biosensing and gene expression profiling.

Single-wall carbon nanotubes (SWCNTs) could be promising delivery vehicles for cancer therapy. These carriers are generally introduced intravenously, however, little is known of their interactions with endothelial cells, the cells lining vessels and mediating clearance of nanoparticles. Here we show that SWCNTs of 1 to 5 μm in length, both “pristine” and functionalized by oxidation, had limited toxicity for endothelial cells in vitro as determined by growth, migration morphogenesis, and survival assays. Endothelial cells transiently took up SWCNTs, and several lines of data indicated that they were associated with an enhanced acidic vesicle compartment within the endothelial cells. Our findings of SWCNT interactions with endothelial cells suggest these may be optimal vehicles for targeting the vasculature and potential carriers of anti-angiogenic drugs. The implications on their biological activity must be taken into account when considering the use of these nanoparticles for therapeutic delivery of drugs. From the Clinical Editor Interactions of single walled carbon nanotubes (SWCNTs) with endothelial cells following IV administration remains unclear. Functionalized and naive SWCNTs of 1-5 mm in length had limited toxicity to endothelial cells in vitro. Endothelial cells transiently took up SWCNTs and were associated with an enhanced acidic vesicle compartment within the cells. These findings suggest that SWCNTs may be promising vehicles for targeting the vasculature and potential carriers of anti-angiogenic drugs.

Recently, nanopore technology has been introduced for genome analysis. Here we show results related to the possibility of preparing “engineered solid state nanopores”. The nanopores were fabricated on a suspended Si 3 N 4 membrane by Focused Ion Beam (FIB) drilling and chemically functionalized in order to covalently bind oligonucleotides (probes) on their surface. Our data show the stable effect of DNA attachment on the ionic current measured through the nanopore, making it possible to conceive and develop a selective biosensor for gene expression profiling.

Point defect formation and stabilization properties, as well as their peculiar spectroscopic characteristics, locally modify the optical properties of insulating materials. Thin layers containing high concentrations of colour centres, hosted in a LiF single crystal and/or a polycrystalline matrix, offer the opportunity to develop innovative light-emitting photonic devices. Control of all the critical parameters should be required on spatial dimension comparable with the optical wavelengths. Recent developments in laser technologies, electron and particles beam methods, and novel photon sources, have opened a wide range of opportunities. An overview of the most significant advances in this field is provided, with particular emphasis on colour-centre LiF-based innovative miniaturised light sources, optical amplifiers and lasers. (© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

We demonstrate that a combination of ion sputtering and soft lithography is an alternative and effective way of nanostructuring soft matter. We create self-organized nanoscale structures on a glass template by irradiating the surface with a defocused, low energy Ar ion beam. Capillary force lithography is then used to transfer the pattern, exploiting the glass transition of polymeric layers. In particular, we demonstrate the pattern transfer of a periodic 150 nm ripple structure onto an organic compound. This new, unconventional combination is then a low-cost strategy that opens the way to a variety of applications in the field of organic-based devices.

We present data concerning the electrical properties of a class of biosensor devices based on bio-functionalized solid state nanopores able to detect different kinds of interactions between probe molecules, chemically attached to the pore surface, and target molecules present in solution and electrophoretically drawn through the nanometric channel. The great potentiality of this approach resides in the fact that the functionalization of a quite large pore (up to 50-60 nm) allows a sufficient diameter reduction for the attainment of a single molecule sensing dimension and selective activation, without the need for further material deposition, such as metal or oxides, or localized surface modification. The results indicate that it will be possible, in the near future, to conceive and design devices for parallel analysis of biological samples made of arrays of nanopores differently functionalized, fabricated by standard lithographic techniques, with important applications in the field of molecular diagnosis.

In recent years there has been a rapid increase in nanotechnology applications to medicine in order to prevent and treat diseases in the human body. The established and future applications have the potential to dramatically change medical science. The present paper will give a few examples that could transform common medical procedures.