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Application of Microarrays Technology to organ transplantation in human with the aid of statistical and non-statistical analysis, including interaction-based methods, is reviewed here.

Biological Faculty, Lomonosov Moscow State University (MSU), Leninskie gory 1, Moscow 119234, Russia Department of Physics, University of Osnabruck, Barbarastr 7, Osnabruck 49076, Germany Department of Health Sciences (DISSAL), University of Genoa, Via Antonio Pastore 1, Genoa 16132, Italy Laboratories of Biophysics and Nanotechnologies (LBN), Department of Experimental Medicine (DIMES), University of Genoa, Via Antonio Pastore 3, Genoa 16132, Italy Nanoworld Institute Fondazione EL.B.A.Nicolini (NWI-FEN), Largo Redaelli 7, Pradalunga, Bergamo 24100, Italy

Nucleic Acid Programmable Protein Arrays utilize a complex mammalian cell free expression system to produce proteins in situ. In alternative to fluorescent-labeled approaches a new label free method, emerging from the combined utilization of three independent and complementary nanotechnological approaches, appears capable to analyze protein function and protein-protein interaction in studies promising for personalized medicine. Quartz Micro Circuit nanogravimetry, based on frequency and dissipation factor, mass spectrometry and anodic porous alumina overcomes indeed the limits of correlated fluorescence detection plagued by the background still present after extensive washes. This could be further optimized by a homogeneous and well defined bacterial cell free expression system capable to realize the ambitious objective to quantify the regulatory protein networks in humans. Implications for personalized medicine of the above label free protein array using different test genes proteins are reported.

677 ISSN 1743-5889 Nanomedicine (2010) 5(5), 677–682 10.2217/NNM.10.46 © 2010 Future Medicine Ltd “Label-free NAPPA technology, in combination with protein nanobiocrystallography and its possible future development using anodic porous alumina along with a cell-free expression system ... appear to form a single approach capable of effectively solving the numerous problems still present in medical diagnosis and therapy.”

This review presents the status of technological developments of nanogenomics and its applications to medicine. Even if particular emphasis is placed on what has been accomplished in our laboratory in the last few years in the area of genes microarrays, significant reference to the recent activity of numerous other groups can be found in Refs 1,2.

This book presents an overview of the current state of research in the field of nanotechnologies and biotechnologies for energy, environment, electronics, and health, as emerging from leading laboratories worldwide. It presents and describes in detail the recent research results in the most advanced nanobiotechnology-based methods and their possibl

Climate change, need for alternative energy and increasing asteroids in size and number represent key issues yielding growing challenges that must be tackled with more determination by the world community. The meeting set in Paris for December by the United Nations represents an unique opportunity to outline a common strategy for them as here outlined.

(2004). Nanocrystallography: an emerging technology for structural proteomics. Expert Review of Proteomics: Vol. 1, No. 3, pp. 253-256.

The photovoltaic (PV) solar cell, converting incident solar radiation directly into electrical energy, today represents the most common power source for the Earth-orbiting spacecraft, and the utilization of organic materials in this context is here explored in comparison with the present state of the art placing emphasis in organic nanotechnology. Poly[3-3'(vinylcarbazole)] (PVK) was synthesized by oxidative polymerization with ferric chloride of N-vinylcarbazole. The resulting polymer was then deposited on solid support by using the Langmuir-Schaefer (LS) technique. The pressure-area isotherm of PVK revealed the possibility of compact monolayer formation at the air-water interface. Different layers of PVK were doped with iodine vapors. The cyclic voltammetry investigation of PVK-doped I/sub 2/ showed a distinctive electrochemical behavior. The photoinduced charge transfer across a donor/acceptor (D/A) hybrid interface provided an effective method to study the PV properties of the composite LS films. The results are compared with other approaches within the biological framework, such as bacteriorhodopsin (BR), and organic nanostructured materials.

The aim of the presented work was to realize and to test hybrid capacitor elements by using of easy and low-cost fabrication techniques. Capacitors were fabricated by using solid solutions of Li salts in poly(ethylene oxide) (PEO), deposited by solution casting. Electrodes were realized from different materials. The electrical tests of the elements were performed in order to check the stability of properties in time and the performance reproducibility.

We present the DNA analyzer (DNASER), a novel bioinstrumentation for real-time acquisition and elaboration of images from fluorescent DNA microarrays. A white light beam illuminates the target sample allowing the images grabbing on a high sensibility and wide-band charge-coupled device camera (ORCA II-Hamamatsu). This high-performance device permits to acquire images faster and of higher quality than the traditional systems. The DNA microarrays images are processed to recognize the DNA chip spots, to analyze their superficial distribution on the glass slide and to evaluate their geometric and intensity properties. Differently form conventional techniques, the spots analysis is fully automated and the DNASER does not require any additional information about the DNA microarray geometry. The DNASER hardware and software architecture is illustrated. Preliminary results are shown from experiments performed on real DNA samples.

Crystallization is a highly demanding and time-consuming task that causes a real bottle-neck in basic research. Great effort has been made to understand the factors and parameters that influence this process and to finely tune them to facilitate crystal growth. Different crystallization techniques have been proposed over the past decades, such as the classical vapor hanging drop method, its variant the sitting drop method, dialysis, cryo-temperature, gel, batch, and the innovative microgravity (space) techniques like free interface diffusion (FID) and counter-ion diffusion (CID). Here, we present a review of the strategies utilizing Langmuir-Blodgett (LB)-based nanotechnologies, and microgravity techniques for obtaining optimal high-quality crystals, as proven by molecular dynamics (MD) and bioinformatics approaches, namely using a clustering algorithm and protein alignment.

A novel method for the formation of ultrathin polycrystal semiconductor layers based on the Langmuir-Blodgett technique is reported. The proposed approach allows the synthesis of layers of pure compounds belonging to the II–VI groups such as CdS and PbS with a thickness resolution of 0·8 nm starting from Langmuir-Blodgett films of fatty acid salts exposed to an atmosphere of hydrogen sulfide. The layers formed are characterized by Brewster angle microscopy on the solid substrate showing good uniformity and constant thickness. This new method could have interesting applications in the formation of thin sensitive layer transducers for enhanced performance field-effect based biosensors such as ChemFET or ISFET.

Nanometer size semiconductor (PbS) particles were synthesized in the fatty acid matrix by exposing the fatty acid salt Langmuir-Blodgett films to an atmosphere of H 2 S. Realization of the structure: electrode-tunnelling gap-particle-gap-STM tip allowed one to register monoelectron phenomena, such as Coulomb blockade and Coulomb staircase. Synthesis of the nanoparticle at the tip of a metal stylus allowed one to avoid the necessity of using STM and permitted one to register monoelectron characteristics using only a feedback in one direction perpendicular to the flat electrode plane. Another phenomenon connected with negative resistance regions in voltage/current characteristics was observed just by varying the tip-flat electrode distance. It was found that bulk properties of the particle material do not play a crucial role in the appeatance of the monoelectron properties.

The recent application of Mass Spectrometry (MS) to Nucleic Acid Programmable Protein Array (NAPPA) technique for proteins identification by non-classical methods leads to the needs of more sophisticated algorithm for peak recognition. NAPPA technique allows for functional proteins to be synthesized in situ directly from printed cDNAs but faces the difficulty generated by the presence of master mix and lysate molecules peaks appearing as background in the overall spectra. A wide range of tools are available to analyze proteins conventional mass spectra corresponding to few molecular species. None of them is optimized for background subtraction. Moreover, peak identification is performed by statistical analysis on characteristics peaks and thus background subtraction can alter outcome by erasing characteristic peaks. A first attempt to overcome the so far discussed problem is here discussed. The result of this effort is the development of SpADS: Spectrum Analyzer and Data Set manager-an R script for MS data preprocessing-therein discussed. SpADS provides useful preprocessing functions such binning and peak extractions, as available tools, and provides functions of spectra background subtraction and dataset managing. It is entirely developed in R, thus free of charge. A cluster k means implementation is here used to improve results of SpADS preprocessing on test datasets and on NAPPA expressed proteins.

We studied laser-microdissection of standard and Langmuir-Blodgett (LB) nanotemplate protein crystals in glycerol solution. The time required for microdissection was significantly longer for LB-crystals as compared to standardcrystals which also more rapidly dissolve. Microfragmentation of lysozyme crystals was observed after extended solvent exposure. Synchrotron radiation nanobeam mapping allowed localizing and aligning cryofrozen lysozyme microfragments. 3D data-sets obtained from two microfragments were refined to atomic resolution. The well-defined electron density maps showed no evidence for damage of radiation of sensitive side-groups. Our results suggest applications of laser-microdissection techniques in structural studies on crystals with a high mosaicity. They also provide a new window for the characterization of protein crystal organization down to the submicron scale, pointing to a new emerging biophysical technique.

Nanogenomics, being the interplay of nanobiotechnologies and bioinformatics, is emerging as an intriguing approach in the field of nowadays biomedicine. Microarrays can produce a wealth of data and details, but they need an algorithm for data reduction to be clearly understood and exploited. The Leader Genes approach, integrating the different available databases and genomics tools, enables the user to search for genes linked to a disease or a cellular process, and to visualize the class of the most important genes, that is to say those having the highest number of interconnections. In this manuscript, we will review the algorithm which has been validated with both experimental and clinical studies. We will describe the different steps that lead to its final version, and we will discuss future perspectives and developments.

The design, realization, and test performances of an electronic junction based on single-electron phenomena that works in the air at room temperature are hereby reported. The element consists of an electrochemically etched sharp tungsten stylus over whose tip a nanometer-size crystal was synthesized. Langmuir-Blodgett films of cadmium arachidate were transferred onto the stylus and exposed to a H2S atmosphere to yield CdS nanocrystals (30-50 angstrom in diameter) imbedded into an organic matrix. The stylus, biased with respect to a flat electrode, was brought to the tunnel distance from the film and a constant gap value was maintained by a piezo-electric actuator driven by a feedback circuit fed by the tunneling current. With this set-up, it is possible to measure the behavior of the current flowing through the quantum dot when a bias voltage is applied. Voltage-current characteristics measured in the system displayed single-electron trends such as a Coulomb blockade and Coulomb staircase and revealed capacitance values as small as 10(-19) F.

Several silicon-based biosensors have been developed for various applications, such as enzymatic and immunological activity determination and cell-metabolism detection. This work describes an electrochemical characterization of ion-sensitive field-effect transistor (ISFET) and light-addressable potentiometric sensor (LAPS) transducers and a test of such devices as detectors of 3T6 (Swiss albino mouse embryo, fibroblasts) metabolism. In particular, our investigation is devoted to some fundamental parameters of these transducers (e.g., pH sensitivity, drift, temperature dependence of the output signal, speed of response) for the purpose of comparing their performance related to cell-metabolism evaluation. The final goal is therefore to analyze the capabilities of these silicon-based transducers for use in a biosensing system for cell-metabolism detection.

A method for preparing samples suitable for calibrating scanning probe microscopes (SPM) and for eliminating any distortions in images is described. Samples consist of polystyrene particles organized in monolayers and bilayers with hexagonal‐ordered domains. The monolayer is not uniform, but is characterized by areas without particles. These discontinuities allow the measurement of the thickness of the monolayer in order to calibrate the z axes, while the lattice constant of the domains can be used as a calibration standard for the x and y axes. The nondeformability of the particles after the deposition on the substrate has been studied by an optical microscope, equipped for interferometric measurements, scanning force microscopy, and scanning tunneling microscopy. The use of these standards directly as substrates for samples is proposed to correct the distortions in the SPM images.

A significant upgrading of a recently introduced biosensor system, called potentiometric alternating biosensor is presented. The transducer consists of a light‐addressable silicon chip, which provides regions properly modified and functionalized to yield sensitivity to either pH or redox potentials. The computer controlled system drives an array of light sources, allowing two‐dimensional signal acquisition and processing. The system can provide multiparameter information related to the local modifications made on the sensing surface of the transducer. This work presents a detailed description of the measuring principles and the spatial resolution obtainable with the system. In addition, the complete circuit design is presented, both for signal conditioning, and for the bidimensional matrix addressing. A typical experiment for selective measurements is presented.

STM images of 10.7 nm dendrimer microspheres were obtained in order to provide a model study of STM imaging of large insulating objects. Negative contrast in these images, observed before only on biological samples, demonstrates that this phenomenon cannot be explained by the biological specificity of a specimen. Large insulating objects may appear as depressions on the constant current STM image due to the interplay of electronic and mechanical interactions between the tip, the substrate and the adsorbate. Utilization of macroscopic elasticity constants in a model of three elastic spheres gives a good correspondence with the reported experimental data. This model can be modified in future in order to obtain microscopic mechanical properties of nm-size objects in STM experiments.

Calibration of scanning probe microscopes (SPM) for atomic (molecular) resolution scans can be carried out on crystalline surfaces. However, SPM scans with atomic resolution are often affected by drift and hence would give false calibration factors. We propose a method which allows to calibrate the SPM instrument eliminating the effects of drift in a first‐order approximation. Scans of the same surface are taken at different speeds and a linear regression is applied to the calibration factors calculated for each scan speed. Applying this method we succeeded in calibrating a commercial SPM system for atomic resolution scans with a precision of better than 2%.

In order to explore the possibility that the assignment of University chairs could be automated, we have carried out a comparative analysis of the relative scientific and technological level of candidates competing nationally for given chairs of first (full professor) and second (associate professor) level, using indicators such as the number of publications and citations in international journals, the number of patents and inventions and few others. This study, contrary to some gloomy opinions, has suggested that at least for what concerns hard science, performances can be measured impartially at least to some extent. Bibliometric indicators, when properly weighted., appear to be effective parameters to monitor degree of excellence in scholastic rating and to establish reliable objective standards. Their utilization, however, to automate the assignment of university chairs appears still questionable and should be limited to the needed provision of computer-assisted selection criteria and reference databank.

The authors outline a method which uses differentially polarized light scattering to study the properties of bacterial cell suspensions, i.e., spores of Bacillus Subtilis. The identification of bacterial cells of different strains, with and without plasmid insertion, was performed by means of differential polarization light scattering (DPLS). The samples displayed distinct angular behaviors for the S/sub 14/ and S/sub 34/ normalized scattering parameters of the Mueller matrix depending on the strain and on a plasmid insertion in the chromosomal unit. These experiments, performed blindly, point out the possibility of achieving real time identification of micro-organisms by DPLS spectrometry. >

At the first C.E.C. Workshop, in Brussels on 28–29 November 1991, attended by over 70 leading European scientists and industrialists, bioelectronics was defined as the ‘the use of biological materials and biological architectures for information processing systems and new devices’. At the end of the Frankfurt Workshop, bioelectronics, specifically bio-molecular electronics, was described as ‘the research and development of bio-inspired ( i.e. self-assembly) inorganic and organic materials and of bio-inspired ( i.e. massive parallelism) hardware architectures for the implementation of new information processing systems, sensors and actuators, and for molecular manufacturing down to the atomic scale’. The subject of this overview is to summarize some of the most significant progress in bio-molecular electronics from neural VLSI networks and bio-molecular engineering. As an example of one possible route, emphasis is placed on the results recently obtained within this laboratory.

A complete potentiometric alternating biosensor system (PAB) is described which utilizes a newly developed transducer based on the light addressable chemical sensor (LAPS) technology, following detailed theoretical optimizations of the relevant physical parameters. Our device, entirely designed and realized ‘in house’, combines measuring chambers with specific electronic cards and the related software programs; the system is completely automated and driven by a personal computer. The electronics was designed in order to obtain a good general signal-to-noise ratio, hence ensuring reproducible results. The software package consists of low-level programs (written both in C and in Assembler) to interact with the data acquisition and controlling cards, and of high-level programs (written in C) to get, display, save and print data files, and to present a suitable user interface. A very important peculiarity of our system is the possibility of connecting different measuring chambers to the same device. This allows the user to perform a wide variety of experiments, utilizing different biological elements, by simply changing the reaction chamber and selecting the appropriate acquisition software. The system has been shown to operate with living cells as well as with enzymes and antibodies. In addition, a particular chamber containing a gold-evaporated transducer can be connected to the main system, allowing redox potential measurements. The system utilizes a fast information recovery from the transducer output signal, and can acquire quantitative data every few fractions of a second. This feature can be utilized to monitor fast acidification or redox processes.

A state-of-the-art review of the role of the Langmuir-Blodgett nanotemplate on protein crystal structures is here presented. Crystals grown by nanostructured template appear more radiation resistant than the classical ones, even in the presence of a third-generation highly focused beam at the European Synchrotron Radiation Facility. The electron density maps and the changes in parameters such as total diffractive power, B-factor, and pairwise R-factor have been discussed. Protein crystals, grown by the Langmuir-Blodgett nanotemplate-based method, proved to be more radiation resistant compared to crystals grown by the classical hanging drop method in terms of both global and specific damage.

An analysis of two‐dimensional fast Fourier transform from images of periodical lattices (like highly oriented pyrolytic graphite) has been performed to understand and decouple the various parameters which account for distorted images in stylus microscopy. The effects of the various sources of image distortion have been described by means of linear maps and a mathematical approach has been developed to find the various correction coefficients resulting from the Fourier space analysis which restore the correct geometry of the images. Furthermore, the trend analysis of the distortion angle upon the scanning frequency shows the possibility of decoupling the role of ‘‘static’’ and ‘‘time dependent’’ distortion parameters. This possibility may be used for an a priori prediction of possible distortions in stylus microscopy and thus for a real‐time correction of the images during scanning.

Measurement of the ac photocurrent in metal/insulator/semiconductor capacitors can be used as a tool to measure minority‐carrier diffusion and lifetime. The amplitude of the ac photocurrent generated at a silicon surface biased into inversion depends on the number of excess minority carriers present at that surface. By comparing this amplitude when intensity‐modulated light is directed to each side of the same device, minority‐carrier diffusion from the back to the front of the device can be characterized. An analytical model of this transport process predicts the dependence of the ac photocurrent on frequency and wafer thickness, and allows the determination of a value of the bulk lifetime free of the influence of surface recombination. Measurements under low‐light intensity levels are presented on n‐type silicon wafers with lifetimes in the 10–100 μs range. Lifetimes are found about a factor of 2 lower than those measured with noncontact photoconductive decay, at high‐light intensity levels. This is exp...

DNA microarrays are one of the most promising methods for molecular genomics, but this technique is often associated with experimental complications and difficulties in the analysis. Moreover, the greatest part of genes displayed on an array is often not directly involved in the cellular process being studied. Recently, we proposed a data mining algorithm, based on the identification of genes involved in a given process, the calculation of interactions among them and their ranking according to number of interactions. Genes in the highest cluster are defined as "leader genes". These findings may lead to an ad hoc and therefore more significant experimentation. However, at present this complex process is performed manually. In this work, we present the general architecture of LeaderGene, an automated tool for ab-initio molecular genomics. Three different and independent parts: (1) Identification of gene list; (2) Calculation of weighted number of links; (3) Genes clustering. Initial inputs are provided by user; then, output of part 1 and part 2, respectively, become inputs of parts 2 and 3. The development of an user-friendly software capable to automatically compute leader genes in a given cellular system will allow further progresses in this field of molecular genomics.

Biosensors are analytical devices which combine a biologically sensitive element with a physical or chemical transducer to selectively and quantitatively detect the presence of specific compounds in a given external environment. The interaction of the compound (the analyte) and the biological element generates a signal (electrical, optical or mechanical) which is measured by an optical or electronic system, usually following amplification. Increasingly, biosensors are exploiting state-of-the-art technologies such as protein engineering and advanced microelectronics to offer easy-to-use, widely applicable devices which are compact as well as cheap.

Alternating photocurrent measurements with light-addressable potentiometric sensors (LAPSs) have been used to monitor pH, redox potential, and ionic concentrations at discrete locations in an electrolyte in contact with LAPS devices. We report here the results of AC photocurrent measurements with insulated semiconductor LAPS devices where the semiconductor either is illuminated through the insulator (frontside) or alternatively from the opposite side (backside). Such comparative AC photocurrent measurements were made with semiconductors of varied thickness, at varied frequency of light intensity modulation, and at several different photoexcitation wavelengths. The results are fit to a theoretical expression which predicts the dependence of photocurrent on modulation frequency, wafer thickness, bulk minority carrier lifetime, and surface recombination velocity. The results are useful to optimize the design of LAPS devices with regard to these parameters. The results also predict optimal conditions for minimal lateral spacing of adjacent sensing areas in LAPS devices.

Nucleosomes, chromatin and nuclei, extracted from rat hepatocytes, were studied by an experimental configuration which measures circular intensity differential scattering (CIDS) and other elements of the polarized light scattering matrix. The Mueller matrix elements, S/sub 14/ and S/sub 34/, that are related to the geometric parameters of the superhelical arrangement of polynucleosomes point to the existence of a quarternary structure at low ionic strength for chromatin prepared by the cold-water method, which is lost by shearing, and is not found in the soluble chromatin prepared through the nuclease method. Only the addition of salt to produce a final concentration of 5 mM MgCl/sub 2/, 150 mM NaCl and 10 mM Tris HCl (pH 7) yields a sizable (S/sub 14/+S/sub 34/) signal in the latter chromatin. This signal is however still different from the corresponding signal of native nuclei and of cold-water chromatin. The (S/sub 14/+S/sub 34/) signal from isolated nucleosomes in consistently very low (nearly zero) as predicted by multiple dipole simulation within the framework of classical electrodynamics. Results are discussed in terms of the topological constraints present in the native long chromatin fiber, which are lost after limited nuclease digestion and after shearing. >

A method for acquiring and processing 3D information from biological specimens imaged with an optical microscope is presented. It is physically based on optical sectioning microscopy, and utilizes a set of digital filters implemented for deblurring optical section images. Rather than the provision of an exact solution for the problem, we have found of more practical use a new approximate method that significantly improves the situation, within reasonable cost parameters.

This work describes IMAGO, an integrated bio-imaging system developed in our laboratory. The whole system consists of a personal computer, a commercially available frame grabber directly plugged into a personal computer, video input/output modules, specific hardware for z-axis movement and light shuttering, and a software package. IMAGO is user-friendly, menu driven and enables one to perform image acquisition with different methods: optical sectioning, flashing epifluorescence, transmitted and phase contrast microscopy. It makes various functions possible, including: image transfer, gray scale processing, conventional and advanced filtering, logical operations, look-up table management, three-dimensional (3D) editing, 3D representation and auto-correlation techniques. More than 100 image processing functions have been implemented and can be easily managed through IMAGO. Examples are given in the area of biophysical research, like 3D representation of nuclei and of electron microscopic images, in situ microscopy of living cells. IMAGO processes information in an x, y, z, t space.

‘A new approach for medical diagnostics and therapy, called ‘nanogenomics’, is emerging from the interplay of bioinformatics and biomolecular microarrays on a previously unseen scale.’

Semiconductor nanoparticles were produced by exposing fatty acid salt Langmuir–Blodgett films to the atmosphere of H 2 S. Particles of different materials, such as CdS, PbS, MgS were formed and studied. Particle aggregation was used for producing thin semiconductor layers and superlattices. Alternating LB films of fatty acid salts with different metals were deposited, varying their thickness. Film structure was investigated by electron microscopy. The measurements revealed that semiconductor superlattices with different alternating layers were formed.

The aim of this work was to realize and characterize rectifying junctions with nanometer thickness based on conducting polymers. Various monolayer films were deposited onto different flat graphite electrodes in order to obtain the Schottky interfaces. The second electrical contact was realized by approaching the monolayer film with sharp tip electrode at nanometric distances, in order to create tunneling barriers. A couple of hundred of junctions were realized following this procedure. The investigated junctions have shown rectifying behavior on the current/voltage characteristics in the 96% of the cases. The analysis of the current/voltage characteristics revealed the typical behavior of Schottky barriers. The ideality factor and the Schottky barrier of the junctions were calculated. Moreover, a linear relationship between the threshold voltage and the tunneling barrier width was revealed. In our knowledge, this is the first report of a Schottky junction realized with a monolayer of polymer film.