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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 possible application of a novel double-iterated Kalman filter (DIKF) as an algorithm for molecular structure determination is investigated in this work. Unlike traditional optimization algorithms, the DIKF does not exploit experimental nuclear magnetic resonance (NMR) constraints in a penalty function to be minimized but used them to filter the atomic coordinates. Furthermore, it is a nonlinear Bayesian estimator able to handle the uncertainty in the experimental data and in the computed structures, represented as covariance matrices. The algorithm presented applies all constraints simultaneously, in contrast with DIKF algorithms for structure determination found in literature, which apply the constraints one at a time. The performances of both paradigms are tested and compared with those obtained by a commonly used optimization algorithm (based on the conjugate gradient method). Besides providing estimates of the conformational uncertainty directly in the final covariance matrix, DIKF algorithms appear to generate structures with a better stereochemistry and be able to work with realistically imprecise constraints, while time performances are strongly affected by the heavy matricial calculations they require. © 1996 by John Wiley & Sons, Inc.

This work describes a novel approach to the realization of ordered protein films. The adopted strategy for the deposition of cytochrome c films involves the synthesis of a compound able to selectiv...

Molecular exchange kinetics between a monolayer of antibody molecules formed on the air−water interface and the protein solution was studied by means of fluorescent labeling. It was shown that ther...

This paper describes the optimal implementation of three newly conceived sensors for both health and environmental applications, utilizing a wide range of detection methods and complex nanocomposites. The first one is inorganic and based on matrices of calcium oxide, the second is based on protein arrays and a third one is based on Langmuir-Blodgett laccase multi-layers. Special attention was paid to detecting substances significant to the environment (such as carbon dioxide) and medicine (drug administration, cancer diagnosis and prognosis) by means of amperometric, quartz crystal microbalance with frequency (QCM_F) and quartz crystal microbalance with dissipation monitoring (QCM_D) technologies. The resulting three implemented nanosensors are described here along with proofs of principle and their corresponding applications.

Langmuir-Blodgett (LB) technology was used to build a high-sensitivity enzyme-based biosensor for medical purposes. Recombinant fungal laccase from Rigidoporous lignosus, as previously described, was used to catalyze a widely used antidepressant in a micromolar range, namely, clomipramine. The topological properties of the laccase thin film were characterized via LB π-A isotherm and AFM (mean roughness 8.22 nm, compressibility coefficient 37.5 m/N). The sensitivity of the biosensor was investigated via UV spectroscopy, and linearity was found in the absorbance peak shift at 400 nm at drug concentration varying up to 20 uM. The enzyme kinetics was subsequently investigated with potentiometric and amperometric measurements, and we found electronic transfer of at least 1 electron, k(s) 0.57 s(-1), diffusion coefficient 3 × 10(-6) cm(2)/s, K(cat) 6825.92 min(-1), K(M) 4.1 uM, K(cat)/K(M) 2.8 × 10(7) mol(-1) s(-1), sensitivity of 440 nA/uM, maximum velocity 1706.48 nA/s, and response time less than 5 s. The amperometric and potentiometric measurements were repeated after a month, confirming the stability of the biosensor.

A STM study of ultra-small cadmium sulphide particles formed in a bilayer of cadmium arachidate exposed to a hydrogen sulphide atmosphere is reported. Particles of nanometre size were identified as negative features inside the film after the reaction. Voltage-current characteristics of particles of different apparent sizes were measured by means of the microscope probe. The behaviour of the tunnelling current indicates the occurrence of a Coulomb staircase at room temperature and reveals a new phenomenon which shows the appearance of regions with negative resistance.

In this work the enzymatic activity of an immobilised Langmuir-Blodgett film of glutathione S-transferase (GST) that catalyses the glutathione conjugation to the l-chloro-2,4-dinitrobenzene has been studied. Silicon wafers or glass slices were used as solid supports and their surfaces were modified by means of silanization with 3-glycidoxypropyltrimethoxysilane. This chemical modification of the surface allowed one to bound the enzyme to the support, avoiding detachment during the measurements. A Langmuir-Blodgett trough was used to form the GST film at the air-water interface and the Langmuir-Schaefer deposition technique was adopted to transfer the enzyme film onto the activated support. Nanogravimetry and spectrophotometry were performed to analyse the formed film in order to obtain data on surface density and functional activity respectively. The results underlined a reliability of the deposition and a maintenance of the enzymatic activity of GST after its deposition onto the solid support. The activity of GST film related to organization, thermal treatment and storage conditions was also studied. The investigation points out that the organisation of the LB film increased the specific activity of the enzyme film with respect to the spread one and that it was maximised when the GST was deposited onto an activated support. The enzymatic activity of GST was still maintained up to 423 K; the immobilised enzyme even showed activity 5 months after its deposition onto the activated substrate and only a small decrease in activity was observed.

Langmuir-Blodgett films of valinomycin appear to be unstable when kept in normal conditions. To prevent recrystallization of the valinomycin, alternating bilayers of this compound and barium salt of disuccinilated glycerol-dialkyl-glycerol tetraether (DS-GDGT) produced from lipid extract of thermophilic archaeon Sulfolobus solfataricus have been deposited. Optical and electron microscopy observations show that the film morphology does not deteriorate for at least three months after deposition. Study of the structure by the X-ray small-angle diffraction technique proves the suggested alternation of valinomycin and lipid bilayers. The period in the direction normal to the substrate plane equals 5.34 nm while the period of the DS-GDGT barium salt multilayer is equal to 3.97 nm. The thickness of the valinomycin monolayer of 0.68 nm is determined from these data. To clarify some structural peculiarities of the prepared films, alternating bilayers of valinomycin and barium behenate have been deposited and studied as well.

The Langmuir-Blodgett technique is applied for depositing thin films of two lipids extracted from the thermophilic archaeon Sulfolobus Solfataricus as well as of a derivative produced by hydrolysis of total lipid extract. These are monosubstituted glycerol-dialkyl-nonitol tetraether with a β-d-glucopyranosyl group (GL-GDNT), bisubstituted glycerol-dialkyl-nonitol tetraether with β-d-glucopyranosyl and phosphomyoinositol groups (GL-GDNT-PH) and asymmetric non-substituted glycerol-dialkyl-nonitol tetraether (GDNT). Films with satisfactory morphology are deposited by both horizontal and vertical lift techniques, but the results are not reproducible. To improve the quality of deposition and the uniformity of the film, mixtures of the lipid GL-GDNT and arachidic acid are used. After dissolution of arachidic acid from the multilayer by hexane highly uniform films are obtained. Studies of the monolayers at the air-water interface and of the deposited films show that probably the molecules in the monolayer are in a form of a U with close packing of isopranyl chains.

Langmuir-Blodgett films of monosubstituted glycerol-dialkyl-glycerol tetraether with phosphomyoinositol group extracted from thermophilic archaebacterium Sulfolobus solfataricus are successfully deposited. However, the procedure of film deposition is rather complicated. Reproducibility of the results is not satisfactory. Non-uniform areas are often observed in the films despite the fact that the same optimum conditions of deposition are used. The films do not possess stability in water. To overcome these problems a series of semisynthetic archaeol lipids have been synthesized. The monosuccinilated and disuccinilated glycerol-dialkyl-glycerol tetraethers appeared to be the most promising compounds. Highly uniform films of barium salt of these lipids can be deposited by the Langmuir-Blodgett technique. The multilayers are stable in aqueous solutions. The quality of deposition of disuccinilated tetraether is similar to that of the usual fatty acid salts. The films are studied by optical microscopy, transmission electron microscopy, X-ray small-angle diffraction, and electron diffraction techniques. Contact angles for the deposited films are measured as well. Data obtained from the surface pressure-area isotherms, X-ray diffraction, and contact angle measurements show that the U-shape of molecules is preferred rather than the extended form.

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.

Glutathione-S-tranferase (GST) is an enzyme able to conjugate glutathione to electrophilic molecules such as 1-chloro-2,4-dinitrobenzene or some triazinic pesticides, such as atrazine, with production of protons. The Langmuir-Blodgett (LB) technique was utilized and optimized to obtain stable monolayers of this enzyme. The film was then characterized by means of circular dichroism spectroscopy, nanogravimetry, and spectrophotometry in order to determine the structure, surface density, and functional activity of the enzyme when packed in the monolayers. The functional analysis of the LB film was also conducted as a function of the number of layers and of the temperature. While the kinetic response as a function of layers suggested that only the external layer was active, the film activity as a function of temperature indicated that, as shown earlier for other proteins, GST in a LB film appears to strictly preserve its functional activity and thus its structure up to 423 K. The enzymatic activity was finally tested in solution with the potentiometric alternating biosensor (PAB) system, with the minimum detectable amount of 1-chloro-2,4-dinitrobenzene being 25 μM. The analysis was highly encouraging for a biosensor application, considering that both structure and function appear largely preserved after the deposition.

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.

A new configuration of the biosensing system named potentiomeric alternating biosensor (PAB) is analyzed. The system is based on an Si/SiO 2 /Si 3 N 4 transducer and allows the monitoring of several biochemical events, such as immunoenzymatic reactions and cell metabolism. the utilization of PAB a immunosensor implies the possibility of using a modified Langmuir-Blodgett (LB) technique for the production of an antibody monolayer. In the first stage the activity of an immobilized LB monolayer of an enzyme (urease) is monitored in order to simulate the final step of the immunoenzymatic assay; subsequently, the enzymatic activity of an immobilized monolayer of antigen (MAb) after incubation with antibodies labelled with urease is monitored. Vacuum silanization with glycidoxypropyltrimethoxysilane (GOPTS) is utilized to ensure covalent binding of the protein LB monolayer to a glass substrate. The analysis with PAB of films thus prepared is preceded by characterization of the layer by means of nanogravimetric and spectrophotometric techniques, which yield useful information on the surface density and activity, respectively. Furthermore, the operative lifetime of the enzyme layer within a period comparable to the standard time of an immunoassay has been studied.

Silicion nitride is normally used as a sensitive surface for silicon-based transducers like ISFETs or light-addressable potentiometric sensors (LAPS), because of its capability to sense preferentially H + ions. We have investigated Si/SiO 2 /Si 3 N 4 heterostructures and studied the surface layers via an atomic force microscope (AFM) and a PAB (potentiometric alternating biosensor) system; in particular, the latter method allows the investigation of the temporal behaviour after input pH steps. A suitable experimental set-up has been installed in order to optimize the pH changes in the measuring chamber; data have been acquired corresponding to unit pH steps. The resulting time constants are calculated and the data related to the surface layer images acquired by the AFM. A correlation between different observed responses and the corresponding surfaces characteristics has been established.

Quartz-resonator nanobalances are utilized here as effective transducers capable of determining with high sensitivity the mass changes due to specific protein-protein, antigen-antibody and ligand-receptor binding or self-assembly of functional complexes. The resulting highly sensitive biosensor is based on two quartz resonators (one active and the second used as a reference) and on Langmuir-Blodgett (LB) protein monolayers. The electronics are composed of two separate blocks, one designed to acquire by a personal computer the data coming from the other card, a 24-bit digital counter directly connected to the two oscillators. In the case of immunosensor application, the active and reference oscillators are covered respectively by antibodies specific to a given antigen and by antibodies non-specific to the antigen, in order to disciminate the physical adsorption effects. Deposition of antibody monolayers is performed by the LB technique in a surface pressure range of 20–35 mN m−1 onto gluteraldehyde pre-treated quartz resonators. A thermal treatment of the antibody layer up to 150 °C results in the reorganization of the film, and significantly improves the sensitivity and the properties of the immunosensor.

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%.

The thermal stability and structure of two uniform Langmuir - Blodgett films made of bipolar lipids from archaea have been studied. X-ray diffraction measurements at various temperatures from 20°C to 100°C were performed on these films, during heating and cooling cycles. The Bragg peak position, the integrated intensity and the peak width have been recorded as a function of temperature. Both intensity of diffraction peaks and layer thickness diminish at high temperatures while the correlation length, normal to the multilayer plane, displays some significant changes during the heating cycle. The data suggest the occurrence of some phase transitions. As a final result, the thermal stability of these two bipolar lipids LB-films is quite different and very high for the Phospho 2 LB film, which is stable till 100°C and is very promising for technological applications.

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.

We report the technique for determining the orientation of the IgG molecules in Langmuir films immobilized on a solid substrate. This technique is based on measuring the binding of the secondary antibodies specifically recognising Fab and Fc fragments of immobilized IgG molecules. Specific binding of anti-Fab and anti-Fc rabbit anti-mouse (RAM) polyclonal antibodies to the film of mouse IgG shows strong dependence on surface deposition pressure of the film indicating changes of molecular orientation occurring in the film. At high deposition pressures the amount of anti-Fab specific RAM antibodies bound with the mouse IgG film is much greater than the anti-Fc specific RAM antibodies. This means that IgG molecules of the film are preferentially oriented with their Fab fragments outside. The ratio of molecules oriented in this way to the opposing molecules is estimated to be 3.125. The technique can be considered rather general and suitable for similar studies of any protein 2-dimensional arrays since the antibodies to different parts or fragments of any protein can be obtained.

Langmuir films of IgG formed at the air-water interface and covalently immobilized on solid supports were studied by means of fluorometry and ellipsometry. Dependencies of monolayer molecular density and thickness on surface pressure were determined. The data obtained make possible a model of the film molecular organization and its dependence on the surface pressure. The films are of monomolecular layer character within the entire range of achievable pressures on the air-water interface as well as on the solid supports. The molecular orientation changes with the pressure. In the monolayer on the air-water interface the molecules are positioned with their Fab-Fab-Fc ploane parallel to the surface at pressures below 20 mN/m ; when compressed up to 35 mN/m, they turn by 90° to reach perpendicular position with respect to the surface. Within the range 20 mN/m < π < 35 mN/m the angle of molecular inclination increases monotonically from 0° to 90° with pressure. The pattern of the orientation-pressure behavior for transferred monolayers is almost the same showing that the film molecular structure does not change upon deposition on to the solid activated substrate.

Deposition of uniform fullerene (C60) films is carried out using the Langmuir—Blodgett (LB) technique. As an intermediate stage an LB film from a mixed monolayer of C60 and a specially selected surfactant compound is deposited. The surfactant compound used forms itself into amorphous uniform LB films and C60 molecules distribute homogeneously in this matrix. Another property of the surfactant compound is its solubility in hexane, in which C60 does not dissolve. One-component C60 film is obtained after treatment of the sample with hexane. To achieve this result mixtures of C60 with several compounds were studied and finally one of them was selected. Surface pressure-area isotherms were recorded to evaluate the degree of C60 dissolution in matrix monolayers. Deposited LB films were studied by optical microscopy, electron diffraction and transmission electron microscopy techniques to record changes of the structure and morphology under variation of the conditions and selection of the compounds. One-component C60 monolayers at the air-water interface as well as C60 films transferred with the horizontal lift technique were also investigated for interpretation of the data on mixed multilayers. Some features of the structure of the studied films are discussed.

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. >

The immunological reaction ability of IgG Langmuir-Blodgett monolayers organized by means of a protein A sublayer has been studied by the gravimetric technique. In order to discriminate the effects of molecular orientation and to preserve the native structure of IgG molecules using the protein sublayer, the kinetics of specific and non-specific binding at an immunoglobulin monolayer deposited onto different substrates, such as a silanized aluminium surface and a surface covered by ovalbumin and protein A sublayers, are compared. It is shown that the prevailing effect is molecular orientation. A sublayer of protein A appeared not only to increase the specific sensitivity of the IgG monolayer but to decrease the non-specific binding as well. For this structure the sensitivity of a monolayer of rabbit anti-mouse IgG towards mouse IgG is 10 pM. The sensitivities in the case of a bare metal surface and ovalbumin sublayer are 10 and 100 times less, respectively. Protein A and ovalbumin sublayers are obtained by the Langmuir-Schaeffer technique. The formation of monolayers on the water-air interface and the deposition onto the solid substrate are studied by means of compression isotherms, gravimetric and ellipsometric techniques. It is shown that the solubility of protein A depends on the surface pressure increasing sharply after a certain value of the pressure. It is also shown that protein A can be deposited onto the substrate in the form of dense two-dimensional monolayers.

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.

Past evidence shows that the secondary structure of photosynthetic Reaction Centres is preserved in Langmuir-Blodgett films at temperatures of up to 473 K, and that the corresponding structure in solution is lost at 328 K. This evidence is correlated with findings in this study that the functional activity of Reaction Centres is preserved up to 368 K in Langmuir-Blodgett films but only up to 308 K in dried random smears (non-oriented films). Interestingly, a similar behaviour is displayed by anti-insulin antibodies in Langmuir-Blodgett films. The role of dehydration in the preservation of secondary structure is investigated using the latter protein, and, upon comparison with the role of close packing is found to be a secondary effect. The preservation of antibody functional properties is monitored up to 423 K and unique catalytic properties appear after thermal treatment. This phenomenon is studied by making surface potential measurements and is found to be caused by the increased anisotropy of the antibody layer after thermal treatment. This increased anisotropy, related to the enhanced order of the film due to thermal treatment followed by slow cooling, is confirmed by X-ray analysis. The above evidence is collected in a unitary picture that attributes the cause of protein thermal stabilization to the close packing induced by Langmuir-Blodgett film formation; the decreased conformational entropy is implicated as the initial condition that allows protein recrystallization after thermal treatment.

Background Genes involved in different biological processes form complex interaction networks. However, only few of them have a high number of interactions with the other genes in the network and therefore they may play a major role. In previous bioinformatics and experimental studies, these genes were identified and termed as “leader genes”. In the current ab initio theoretical study, genes involved in human OLP (Oral Lichen Planus) pathogenesis are identified and ranked according to their number of interactions, in order to obtain a broader view of its molecular mechanisms and to plan targeted experimentations. Methods Genes involved or potentially involved in OLP were identified by systematically querying several databases until the identification of a final set of genes. Interactions among these genes were mapped and given a significance score using STRING database. For each gene, significance scores were summed to obtain a weighted number of links (WNL) and subsequently genes were clustered according to this parameter. The genes in the highest cluster were termed as leader genes; the other ones were ranked as class B genes, class C genes, and so on. This study was complemented by a topological analysis of the network, carried out using Cytoscape, BinGO and FANMOD software. Results The interactions in the obtained network showed power law behaviour, in agreement with the scale-free topology theory of the biological graphs. 132 genes were identified and five of them (namely, JUN, EGFR, FOS, IL2, ITGB4) were classified as leaders. Interestingly, all of them but EGFR were up-regulated and were widely distributed in the network (in term of topological parameters such as stress, eccentricity and radiality) and showed higher topological coefficients than the other genes. Conclusions Even with the limitations of any ab initio analysis, this study can suggest targeted experimentation, focused on the leader genes and therefore simpler to be analysed than mass scale molecular genomics. Moreover, it may suggest new potential risk factors and therapeutic targets.

Structural proteomics based on synchrotron radiation [1] and label-free microarrays [2] have recently provided the nanotechnological framework for significant advances in medical diagnosis and therapy [3,4], laying down, at the nanoscale, level the foundation and definition for functional and structural proteomics in nanomedicine [5]. At the same time, with the introduction of new matrices from organic, inorganic and biological nanocomposites [6] many applications in sensor technology to detect biological molecules of medical and environmental relevance have emerged with significantly more selectivity and sensitivity than what had been obtainable so far [7,8]. Of particular interest are the sensor developments originating from the combination of modern functional proteomics [2–4] with quartz crystal microbalance, exploiting both frequency shifts (Df) and dissipation factor (D) for the simultaneous evaluation of mass variation (Sauerbrey equation) and viscoelasticity at the nanoscale [9].

X-ray atomic structure of recombinant Hell's gate globin I (HGbI) from Methylacidophilum infernorum was calculated from the X-ray diffraction data of two different types of crystals: obtained by classical hanging drop and by LB nanotemplate method under the same crystallization conditions. After the accurate comparison of crystallographic parameters and electron density maps of two structures they appears to be quite similar, while the quality of the crystals grown by LB nanotemplate method was higher then of those grown by classical method. Indeed, the resolution of the LB crystal structure was 1.65 A, while classical crystals showed only 3.2 A resolution. Moreover, the reproducibility of this result in the case of LB crystals was much better-nine crystals from 10 gave the same structural results, while only two of 10 classical crystals were appropriate for the X-ray structure resolution.

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.

Self-assembly of polyelectrolytes-polystyrenesulfonate (PSS) and polyallylamine (PAH) with added salts of MnCl and NaBr was studied by x-ray reflectometry and ellipsometry technique. The thickness of PSS-PAH bilayer was measured to be 5.1±0.2 nm according to reflectometry and 6.1±0.7 nm according to ellipsometry. The discrepancy in data is attributed to the difference in the interaction of the interfaces with x-rays and visible light. The films are found to be rather homogeneous and the deposition process regular. The refraction indices of the deposited films were found to ben0=1.50±0.05,k0=0.07±0.05,ne=1.53±0.05ke=0, optical axis being perpendicular to the surface. The values of refractivity characterize the whole film (up to seven bilayers) and do not vary with increasing thickness.

A method of surface modification of chips of potentiometric sensors by 3-glycidoxypropyltrimethoxysilane (GOPTS)1 in gaseous phase for covalent immobilization of antibody Langmuir-Blodgett films has been studied. Antibody monolayers are formed at the liquid-gas interface and transferred onto sensor chips at a surface pressure of 25 mN m−1. The immunological activity of immobilized RAM antibodies is determined on the basis of specific binding of enzyme-labelled antigen. The binding of HRP-labelled RAM antibodies to the immobilized antigen monolayer is estimated by using the conventional spectrophotometric method. The enzymatic and immunological activities of different components of the ‘sandwich’ scheme of the antigen detection have been characterized.

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.

Native chromatin has been characterized at different ionic strengths, according to different methods of preparation, by means of circular dichroism and ethidium bromide intercalation. For a more precise interpretation of these results, a series of analogous experiments on phagic DNA, as a function of its circularization, and on mononucleosomes have been performed. Results are discussed here in terms of high order chromatin structure, DNA supercoiling, and topological constraints.

Langmuir-Blodgett monolayers of photosynthetic reaction centres from Rhodobacter sphaeroides have been studied by scanning tunnelling microscopy. Freshly deposited films were studied both in the dark and in the light. In the dark, images revealed molecular structure with 64 A and 30 A periodicities, which correspond to protein and sub-unit sizes known from X-ray crystallography, while no periodic structure appeared in the light due to the tip action on the excited proteins. STM voltage-current measurements showed the charge separation in single protein molecules in the film and their different behaviour in the dark and light. Together with surface potential measurements at the macroscopic level, they indicated the preservation of reaction centre activity in the monolayer. By fixing the protein layer with glutaraldehyde, it was possible to prevent the perturbing tip action and obtain a periodic molecular structure with 30 A spacing even in the light. After heating at 150°C, the unfixed film reorganized itself into a long-range ordered state with a hexagonal structure of 27 A spacing but with no activity.

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...

Based on the existing technology of light addressable potentiometric sensors we developed a system for measurements of cellular acidification. The system is able to monitor the pH variations of a micro environment where living cells are maintained, fixed on a coverslip; the acquired signal is proportional to the acidic products of cell metabolism. Our study is devoted to estimating the limits of acidification for which the viability of mammalian cells is still maintained. As the system basically consists of a micro volume flow chamber, it is possible to continuously refresh the cell medium, or to stop the flow, so causing acidic metabolites to build up. In a flow-off condition cells are damaged, as long as the flow halt situation is maintained, as previously shown. The experiment consists of a set of measurements performed alternating flow-on and flow-off steps of increasing time duration. By means of such experiments we also estimated the number of H + ions derived from the acidic metabolic products secreted by the cells.

The structure of the immobilized Langmuir-Blodgett films of IgG and their immunological activity were studied by means of ellipsometry. The dependence of the film thickness on the surface pressure of deposition provides evidence of the tilting of molecules with an increase in the pressure. Below pressures of 30 mN m −1 the thickness of the film is approximately 4 nm which coincides with the smallest dimension of the IgG molecules. At pressures between 30 and 40 mN m −1 the thickness increases sharply achieving a value of about 10 nm which is equal to the largest molecular dimension. A further increase of pressure does not show in terms of growth of the thickness. This means that the films are transferred from the water-air interface in the form of a 2-D ordered monomolecular layer. The dependence of the immunological activity on the pressure of deposition was shown to have a descending pattern. Different mechanisms are proposed which explain the decrease of the immunological activity of the IgG molecules in the film with an increase of the surface density such as the blocking of the active sites and the decrease of the conformation mobility of the Fab fragments.

Reversed micelles of sodium diisooctylsulfosuccinate with cytochrome c were used as spreading solutions for Langmuir-Blodgett film formation. A condensed monolayer with closely packed surfactant molecules in the air and closely packed protein molecules under the water surface was formed after several repetitions of the compression-expansion cycle. The resulting film was transferred onto the solid substrate. The films were studied by circular dichroism and by small-angle X-ray scattering. It was shown that the metalloprotein in the film is not denatured and the resulting thin film is significantly ordered, more so than the corresponding monocomponent protein Langmuir-Blodgett films.

Ultrathin films of MEH-PPV/C60 nanocomposite with various C60 content were fabricated onto ITO-coated glasses by means of Langmuir–Schaefer method. The cyclic voltammetric investigation on these ultrathin films suggested that these nanocomposites show distinct electrochemical behaviors at various composition ratios. Keywords: Poly(phenylene vinylene), Fullerene, Langmuir–Schaefer films, Cyclic voltammetry, Photovoltaic cells

Recent findings indicated that the SMILE gene may be involved in kidney graft operational tolerance in human. This gene was found to be up-regulated in blood from patients with a well functioning kidney transplant in the absence of immunosuppression compared to other transplanted recipients with clinically different status. A microarray study of SMILE knock-down and phorbol 12-myristate 13-acetate (PMA) activation in HeLa cells was herein compared to our earlier analysis based on microarray data of kidney allograft tolerance and rejection in humans and in a rat model of allograft transplantation to determine possible new genes and gene networks involved in kidney transplantation. The nearest neighbors at the intersection of the SMILE knock-down network with the human tolerance/rejection networks are shown to be NPHS1 and ARRB2, the former (Nephrin) being involved in kidney podocyte function, and the decrease of the latter (Arrestin β2) being recently shown to be involved in monocyte activation during acute kidney allograft rejection in rat. Moreover, another one of the neighbors at the intersection of SMILE network and tolerance/rejection networks is XBP-1, that we report previously to be increased, at a transcript level, after ER stress in SMILE silenced cells. Finally, in this study, we also show that topological properties (both local and global) of joint SMILE knock-down network—tolerance/rejection networks and joint PMA activation network—tolerance/rejection networks in rat and human are essentially different, likely due to the inherent nature of the gene SMILE and the mitogen PMA, that do not act the same way on genes and do not interfere the same way on networks. We also show that interestingly SMILE networks contain more feed-forward loop (FFL) motifs and thus SMILE calls for a more fine-tuned genetic regulation. J. Cell. Biochem. 113: 1820–1832, 2012. © 2011 Wiley Periodicals, Inc.

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.

Periodontitis is a complex multifactorial disease and is typically polygenic in origin. Genes play a fundamental part in each biologic process forming complex networks of interactions. However, only some genes have a high number of interactions with other genes in the network and may, therefore, be considered to play an important role. In a preliminary bioinformatic analysis, five genes that showed a higher number of interactions were identified and termed leader genes. In the present study, we use real-time quantitative polymerase chain reaction (PCR) technology to evaluate the expression levels of leader genes in the leukocytes of 10 patients with refractory chronic periodontitis and compare the expression levels with those of the same genes in 24 healthy patients.Blood was collected from 24 healthy human subjects and 10 patients with refractory chronic periodontitis and placed into heparinized blood collection tubes by personnel trained in phlebotomy using a sterile technique. Blood leukocyte cells were immediately lysed by using a kit for total RNA purification from human whole blood. Complementary DNA (cDNA) synthesis was obtained from total RNA and then real-time quantitative PCR was performed. PCR efficiencies were calculated with a relative standard curve derived from a five cDNA dilution series in triplicate that gave regression coefficients0.98 and efficiencies96%. The standard curves were obtained using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and growth factor receptor binding protein 2 (GRB2), casitas B-lineage lymphoma (CBL), nuclear factor-KB1 (NFKB1), and REL-A (gene for transcription factor p65) gene primers and amplified with 1.6, 8, 40, 200, and 1,000 ng/μL total cDNA. Curves obtained for each sample showed a linear relationship between RNA concentrations and the cycle threshold value of real-time quantitative PCR for all genes. Data were expressed as mean ± SE (SEM). The groups were compared to the analysis of variance. A probability value0.01 was considered statistically significant.The present study agrees with the preliminary bioinformatics analysis. In our experiments, the association of pathology with the genes was statistically significant for GRB2 and CBL (P0.01), and it was not statistically significant for REL-A and NFKB1.This article lends support to our preliminary hypothesis that assigned an important role in refractory aggressive periodontitis to leader genes.

To the Editor, Much attention has been focused on the attempt to undertake global analysis of protein expression and in particular to analyze the changes in protein expression associated with several processes such as differentiation and cell cycle progression. In general, cell differentiation— or proliferation—requires de novo protein synthesis and/or post-translational modification, in particular phosphorylation of proteins such as transcription factors. Consequently, there is a need to identify the proteins that are being modified, as well as to know when and where in the cell modifications occur. We focused our studies on the Chinese hamster ovary (CHO) mammalian tumor cell line, a good model to study the cell differentiation. It is known that cyclic 30,50monophosphate adenosine (cAMP) transforms CHO cells, in vitro: they lose characteristics associated with malignancy and adopt characteristics of normal fibroblasts (‘‘reverse transformation’’) [3, 7, 9]. Puck et al. [8–10] found that cAMP regulates the structure of the cell cytoskeleton, which in turn strongly influences many differentiation properties including the assumption of normal or cancerous behavior in mammalian cells. In a previous study, we analyzed how CHO-K1 cells display different nuclear protein contents when undergoing differentiation by cAMP [14]; our Mass Spectrometry (MS) investigation revealed a change in the composition of nuclear proteins associated to an inhibition of the protein expression with possible implications for the control of cell reverse transformation [14]. The mechanism of the transformation, however, is as yet obscure and need further exploration in all compartments of cell proteome which we have carried out with this study. Fundamental changes in properties of the cell membrane have indeed been reported but the molecular mechanisms generating these structural changes are largely unknown. The changes in the concentration of the different metabolites and the different protein composition associated with morphological changes observed are also largely unknown [8]. This study wants to explore more deeply the effect of cAMP differentiation on the proteome of CHO cells, considering that the changes in the levels of nuclear envelopeassociated proteins and chromatin-condensing proteins [14] constitutes only the first step toward developing an understanding of cellular mechanisms for controlling nuclear shape. Due to the complexity of eukaryotic proteomes, and the divergence of protein properties, it is beneficial to prepare standardized partial proteomes in order to maximize the coverage of the proteome and to increase the chance to visualize low abundance proteins and make them accessible for subsequent analysis [1]. The strategies used to prepare such a complex proteomic samples for MS analysis involve many steps. For protein extraction we chose to perform a subcellular fractionation to separate the whole cellular proteome in four fractions. In literature, there are several works in which this approach to proteome analysis is adopted [1, 6, 12, 13], but always to analyze the behavior of a restrict number of proteins, for example to investigate changes in subcellular localization of regulatory proteins impacted by experimental or disease parameters. R. Spera C. Nicolini Nanoworld Institute, Fondazione El.B.A. Nicolini, Largo Redaelli 7, Pradalunga, Bergamo, Italy e-mail: rspera@ibf.unige.it

A simple tool for surface density measurements of deposited Langmuir-Blodgett films based on the principle of nanogravimetric piezoelectric resonators has been developed. This device can be very useful for monitoring the quality of the deposited films by checking the functional dependence of the surface density upon the number of deposited layers. Moreover, knowing the molecular weight and the area per molecule of the deposited compound, it is also possible to calculate the packing degree. This parameter can provide a direct method for comparing different techniques of deposition and film formation, taking into account the different behavior of the molecules at the air/water interface and on the solid substrate. The developed tool can, from another point of view, provide information on the area-per-molecule of those samples whose properties do not allow its estimation by usual pressure/area isotherms.

Peak-picking is the lowest-level task of the interpretation of two-dimensional, and multidimensional Nuclear Magnetic Resonance (NMR) spectra in general, for protein structure determination. It consists of individuating peaks on two-dimensional frequency spectra, for further elaboration. The performances of several feedforward artificial neural networks trained with back propagation with temperature on the task of peak-picking are compared. The best one averages less than an approximate 5% error on well-defined spectral regions. The performances of the network are comparable with those of a human expert; the consequences of this fact on the possibility of improving further the performance of the network are discussed.