NWI FEN LABS Science and Technologies

NWIFEN

This section represent a summary of expertise and priorities of th Institute over the years,

It is possible to immobilize protein molecules on solid supports preserving their functional properties in environmentally extreme conditions. For example, Langmuir-Blodgett techniques enable us to maintain the protein secondary structure stability at high temperatures. In the nanoproteomic field technologies such as Langmuir-Blodgett (LB) deposition, monolayer engineering, spreading, self-assembly in electric field, electrodeposition and their combinations are strategic.

During the project nanoproteomics has been employed to address some of the following areas:
Green chemistry has been realized as a necessary alternative to traditional chemical processes. For this reason biocatalytic processes have been improved. However industrial examples of these processes are limited because of the low efficiency of the immobilization techniques employed today. The Nanoworld Institute has been concerned with the realization of new techniques in order to increase the biocatalysts efficiency. One of the promising ways is the application of the so called metho ds of monolayer engineering. Such an approach provides the possibility to utilize monolayers of different compounds for the creation of biocatalytic media with nanometric scale resolution.
Protein nanocrystals will be grown to enhance protein structure determination at atomic resolution by X-ray diffraction and synchrotron radiation. This need is becoming more and more severe with the rapid advances in biotechnology, molecular pharmacology, medicine and material science that require understanding of biological processes at the atomic level. In spite of recent advances in protein crystallography a large number of proteins playing a critical role in living mechanisms have been crystallized so far. Protein crystallization still remains the slowest step in protein structure determination.

Figure Lysozyme crystals grown by LB nanotemplate

Protein crystallization techniques are usually based on the same vapour diffusion methods with different experimental conditions variation. But this approach often has a random and irreproducible success. This is why protein crystallography is often called art instead of science. New methods of protein crystallization based on the thin films techniques make it possible to use the force of nanotechnology in construction of the ordered templates, which allow crystals of proteins to be obtained that have proved elusive using the classical methods.

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Figure X-rays Diffraction of Lysozyme crystals obtained at -30°. Left: Classical Hanging Drop Method. Right: Protein Thin Film Template Method

Nanophotonics and Nanobiosensors

As for the nanophotonics and nanobiosensors, one of the main research objectives of the recent years is realization of nanometer-scale electronic, optoelectronic, and biosensor components and their possible use in various applications. Moreover, considerable interest was demonstrated towards employment of biological materials that are able to operate on molecular level as a single design unit. The possibility to employ new materials and innovative technologies allows to realize conceptually new prototype devices, potentially competitive to the existing in silica technologies.

A large part of commercially available microarrays is manufactured using the photolithographic methodology proposed by Affymetrix. In this case, for DNA chip production, the synthesis of oligonucleotides with the required sequence is carried out step by step directly on the functionalized glass surface. To newly activate the surface, protection must be removed at the specific site, for irradiation of the sample with light through a photolithographic mask. Then, the chemical reaction is repeated with another nucleotide and so on. The in situ synthesis has some advantages compared to deposition of presynthetized oligonucleotides, even if the amount of the synthetized compound (in this case on the surface) is too small to precisely control the sample composition in the array spot. In fact, although particular analyses indicate that high quality of the structures may be achieved, specific equipment is required for quality control. The unavoidable drawbacks result from the structure of the oligonucleotides at the surface “coating”. In contrast, the presynthetized oligonucleotides can be carefully purified and controlled before usage. To produce a microrarray starting from presynthetized oligonucleotides, the surface of glass previously activated is sprayed (spreading) with drops of specific product using robots (arrayers). This system must necessarily be highly precise. These devices are commercially available but they introduce very high cost. The commercially available methods operate at the micron level (it is more correct to speak of “micro”-array), while the ones proposed by the Nanoworld Institute compose a macro research project aiming to realize and characterize methods operating at the “nano” level, with the objective of realizing nanostructures to verify the possibility of moving towards the “nano”-array.

Figure. Microarrays can be monitored by CCD Microscope, now at University of Genova LBN as shown above,  and by an in house built DNASER developed in cooperation with Hamamtsu now in Pradalunga at FEN  as shown below

Polymers

Recent studies have demonstrated the real possibility of performing chemical reactions in the solid state and have revealed advantages in terms of efficiency and selectivity with respect to reactions in solutions. Low mobility of molecules in the solid state suggest that both the reaction rate and its probability will decrease drastically in the absence of solvents. In reality, experiments have demonstrated that molecules can move freely and react, sometimes with surprising yield and kinetics. Moreover, these solid state reactions provide some advantages with respect to solutions, such as: low environmental hazard, simplicity, reduced costs and easier industrialization of processing. Generally, these processes require agglomeration and mixing of the used reagents. This type of the approach is very important at the industrial level concerning the synthesis of molecules and polymers. Currently, great scientific and technological development in the field of organic electronics is directed to the conjugated molecular systems, where conducting polymers (CP) have fundamental and primary role. The production of such systems must often be performed, because of insolubility, in hazardous solvents. An aim of the project is the development of new methods of the polymerisation in the absence of polymer solvents. The innovation of the approach is connected to the direct synthesis of polymers starting from reagents without precursor molecules. This strategy does not forecast, however, classic activation procedures such as thermal conversion and irradiation. The present study is important not only for the basic research in to the understanding and development of solid state chemistry but also in the environmental and industrial applications.

Figure Two different configuration of Atomic Force Microscope built in house in cooperation with Elbatech srl

Carbon Nanotubes

Carbon Nanotubes

The discovery of carbon nanotubes has been attracting considerable attention because of their own unique physical properties and their potential for a variety of applications. CNTs have the following properties:

(1) High length-diameter ratio;
(2) Small curvature radius at the extremities;
(3) High electrical conductivity;
(4) High stability;
(5) Good mechanical strength.

These characteristics make them good materials for “building blocks” of new materials for different applications. The possibility of using CNs in the following applications will be considered:

The use of hydrogen as fuel in alternative of fossil fuels, is attracting due consideration all over the world. The chief reason of hydrogen development consists in fuel cell innovations, which produce electric energy by using hydrogen without polluting the environment. An increasing number of researchers are theorising that structures made up of carbon may be used as hydrogen system storage.

As hollow tubular structure of the size of some nanometers, are used by living form of life, in order to perform different biological functions, CNTs can be used in integrated biological systems and in biomedical devices. CNTs constituted by atoms of carbon, which have conducting properties, are very strong and flexible materials. They can be modified or functionalized by the introduction of side chains, which can provide different chemical-physical properties. These relevant qualities allows to use them in many fields and in medicine seems to be possible to use them as carriers of a wide range of molecules. The angiogenic therapy provides a new approach to revascularization when traditional techniques fail. The mechanism of vascular repair requires endocrine and paracrine activation, and the local delivery of growth factors may stimulate the endothelial response to ischemia. The production of device for the delivery of nano-quantities of active principles at the level of the stenotic vessel will provide a possible solution of the above mentioned problems. On the other side, angiogenesis is the last negative consequence of retinal hypoxia due to diabetes. This very late event may be avoidable only with very early intervention on the pathogenetic chain linking oxidative damage, thrombosis and apoptosis of the microvascular cells.

Figure Small Molecuòle

Inorganic Nanoparticles

Recent years have shown a marked development of the synthesis of metal and semiconductor nanoparticles, stabilized by organic protection, called Monolayer-Protected Cluster molecules (MPC). The nuclear dimensions of these particles can be modulated from a max diameter of about 5 nm, decreasing to sizes less than a nm. The particles have ordered crystal structure and quantized dimensions providing closed atom arrangement (“magic numbers”) tending to reach the equilibrium form of the truncated octahedron. The properties, the materials are between solid crystalline and single molecules, and provide the interesting perspective of displaying new size dependent chemical, electric and physical properties. Gold, silver, or alloys of gold, silver, platinum, palladium and copper can form nuclei of the complexes. Protective organic monolayers are always constituted from molecules with functional thiol group, that are self-assembled at the metal nucleus surface. Alternatively, it is possible to realize semiconductor nanoparticles, such as CdS, PbS etc., also stabilized by organic monolayers. Nanocomposites incorporating such particles will be produced.

Nanoelectronics

The next generation of electronic devices will be based on molecular properties and nanomaterials. In principle, these devices can reach nanometric sizes and demonstrate thermodynamic effects of considerable importance for data processing when compared with existing silicon devices. It is obvious that nanoscale dimensions can be achieved more easily using a “bottom-up” appproach involving self-assembly of molecules rather than lithography. The final step of any fabrication of such systems is connected to the interfacing of formed molecular architectures with the outer world (macroscopic). An aim of the project is the realization of electronic devices and research will include studies of fundamental stages, such as synthesis and design of supramolecular devices, development of the architecture and improved methods of the chemical assembling. Different methods of solving the problem with one common feature – realization of supramolecular architecture – will be assessed. One possible approach is based on the utilization of “interlocked” (electrochemically active) molecules, such as rotaxane or catenane, which can act as electrically controlled molecular switches. Another class of material which can result in the molecular electronic devices is represented by single-walled nanotubes (SWNTs). Interesting chemical and physical properties of SWNTs have led to interest for numerous structural and electronic applications. However, there is a strong limitation of the utilization of the derivatives of these systems due to their intrinsic property. In fact, insolubility of NTs in practically all solvents limit their functionalization. The need to functionalize has resulted in research in the direction of noncovalent functionalization of NT. The noncovalent functionalization represents an interesting opportunity for the attacment of lateral chains to nanotubes, without the destruction of the bond net. Therefore, providing the necessary solubility without changing the electrical properties of the NTs.


LB Through 1

Figure  (above) In house built LB throughs now at University of Genova LBN ; (belowe) MDT-NN LB through in Pradalunga at FEN

The molecular and supra molecular mechanism of gene expression of eucariotic cells are still to be fully understood. It is well known that the gene expression related to cell differentiation, proliferation and other complex functions is under control of many factors with specific time and spatial requirements into cells and tissues. The new techniques of SAGE and DNA microarray, provide powerful tools that will be employed in the study of the coordinate gene transcription occurring during modification of cell function. It will be possible, as an example, to study the modification of gene expression due to genetic diseases such as AT, identifying possible modifications of translation in association with specific mutations of the ATM gene or to study the genomic organization of parasites (such as Leishmania major); they express, during the vital cycle different sets of genes offering valid approach to specific therapies. Another possible application will be to identify the patterns of alterations of gene expression related to thrombosis, apoptosis, cell proliferation/neoangiogenesis of the capillary cells due to diabetes.

Potentiostate

Figure. Protein atomic strucure determination is accomplished with the aid of computer graphics  (above) and Electric properties of enzynes  is determinated by Potentiostat (below), both in Pradalunga at FEN

Figure. Fundamental tool in Proteomics is the shown Mass Spectrometer built by Brooker and acquired by NWI (50% University and 50% Fondazione ELBA NIcolini)  and now still in Genova University Biophysics and Nanobiotechnology Laboratory

The innovative instrumentation developed during previous research provides the qualifying technological platform to the FEN NWI LABS , namely:

1-DNAser instrument, developed within the project BTA9 with Polo Nazionale Bioelettronica and Hamamatsu, constitutes an innovative technology, competitive compared to the available devices from Affymetrix, for microarray analysis in biotechnologies.

DNASER

 

Figure : DNASER Microarrays

2-Evaporator being developed in cooperation with High Vacuum Process for metal deposition.

Device Metal Deposition

In house Evaporator

3.Various instruments for specialized electrochemical analysis using functionalized matrices to measure cholesterol and other compounds of medical relevance, realized within the project BTA7, but also usable as a technological platform for electrochemical measurements in the field of nanotechnologies of nanostructured materials.

AFM Ellipsometer

Figure : AFM (above) Laboratories with Ellipsometry (below on the right)

4.Kinetica Nanogravimeter  now at Genova LBN and AFM microscope now at Pradalunga spawning from research activity included into the “Scientific and Technological Park of Elba” project and made available to the NWI within the presently operating agreement with Elbatech srl.

X-Ray Diffractometer

5.Organic Batteries developed in cooperation with ENEA

6.Organic Photovoltaic Cells for space and energy developed in cooperation with Edison

7.Organic Passive (Capacitors, Resistors) and Active (LED, Diodes, Transistors) components developed in cooperation with ABB

8.Drug Discovery and Protein Crystallization via Nanocrystallography developed in cooperation with Elba Foundation

9.Drug Delivery via Carbon Nanotubes

In Pradalunga at the newly created NanoWorld Institute FEN Laboratories the integration of nanoinstrumentation with office space is shown in the figure in the DNASER area dedicated to microarrays characterization of genes and proteins via fluorescence

Lab Office with Elba Labs view

I

In spite of the participation of industrial companies in research projects of the Institute, the intellectual property of the NWI is guaranteed through ad hoc agreements. In recent years, the research group lead by Professor Nicolini under the umbrella of Elba Nicolini Foundation , and now FEN  NWI LABS, accumulated numerous patents, including international (World Patent Index), European (European Patent Index) and National.

The activities of technological transfer consists of:

relations with the industrial and productive companies at the local, national and international level

connection to the technological transfer of the public institutions

financing by national and international agencies

partecipation to the international research collaborations.