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Our research relates to the implementation of Nucleic Acid Programmable Protein Array (NAPPA) analysis by matrix assisted laser desorption/ionization mass spectrometry (MALDI MS). Such analysis enables interrogation of protein-protein interactions in a labelfree manner by desorption and ionization of analytes (e.g., proteins synthesized from the immobilized cDNA and/or protein bounded to these). Mass spectrometry, in fact, has the unique advantage of being able to determine not only the presence but also establish the identity of a given ligand. However, the development of a MALDI MS-compatible protein microarray is complex since existing methods for forming protein microarrays do not transfer readily onto a MALDI target. Here we present a novel method for implementing a procedure to analyze protein contents on NAPPA protein microarrays using MALDI TOF mass spectrometry.

Protein X-ray crystallography will remain the most powerful method to obtain the protein 3D atomic structures in foreseeable future. However, the production of the protein crystal as well as it quality (order, intensity of diffraction, radiation stability) remains the major problem. Many important proteins including those of life science interest and pharmaceutical industry impact are difficult to crystallize. The second major problem in protein crystallography is radiation damage of obtaining crystals which can only be partially overcome by existing methods. In the present work we use the protein LB nanotemplate crystallization method – generalized procedure for triggering of crystallization of any given protein, which allows to obtain radiation stable and high quality diffracting crystals for further X-ray analysis by synchrotron radiation. We apply LB nanotemplate method to crystallization of L-asparaginase from Rhodospirillum rubrum. This protein has potential application for combined chemical and enzymatic therapy of malignant blood disorders and therefore for new anticancer drug development. We also compare the diffraction quality of asparagines crystal obtained by classical method and LB nanotemplate and report preliminary X-ray diffraction characterization by synchrotron radiation.

Conductometric monitoring of drug-gene and drug-protein interactions is of fundamental importance in the field of molecular pharmacology. Here, we present our main findings and characterizations of an important antiblastic used in neuro-oncology (Temozolomide), interacting with selected proteins that represent predictive biomarkers of the rate survival of the patients, of the outcome of chemotherapy and resistance to drug itself (namely, BRIP1 and MLH1). We use our previously introduced two genes along with previously described Nucleic Acid Programmable Protein Arrays (NAPPA)-based nanoconductometric sensor. We performed a positive control (Temozolomide plus MLH1 protein), a negative control (Temozolomide plus BRIP1 protein) and a multi-gene experiment (Temozolomide plus BRIP1&MLH1 being co-expressed), showing that we are able to properly perform pharmacoproteomics tasks, discriminating each protein and drug unique conductance curve as well as their interactions, even in the presence of multi-proteins being immobilized. Moreover, in the last part of our paper, we used a multiple regression model in order to predict the behavior of Temozolomide when exposed to BRIP1&MLH1 co-expressed and we showed that we are able to predict the drug-protein interaction profile with a good regression coefficient.

Laser-induced microfragmentation of LB nanotemplate-induced protein crystals in glycerol solution resu lts in distinct, coherently diffracting domains. Only c rystals produced according to the Langmuir-Blodgett (LB) nanotemplate technique reveal in all four prot eins being tested (lysozyme, insulin, thaumatin and ribonuclease) domains highly radiation resistant, w hile the crystals produced by the standard hanging drop crystallization method do not. Actually the very sa me laser exposure causes the disappearance of these “classical” protein crystals during the same time f rame of 40 min needed for the laser cutting in all four proteins being tested. The microdiffraction of micr ocrystals prepered by the combination of LangmuirBlodgett and Laser technologies proves that not onl y the Lysozyme survives the process, as shown recently by nanodifraction, but also all three other model p roteins appear to behave similarly well, namely ins ulin, thaumatin and ribonuclease. The result confirms the emerging of a new biophysical technique uniquely usefull for synchrotron radiation studies based on small protein microcrystals uniquely radiation resi stant when prepered by LB nanotemplate and subsequently fragmented by Laser.

1) Construction of SNAP-based Genes Nanoarrays, using gold surface coated for 10 minutes with 2% solution of 3-Aminopropyltriethoxysilane (APTES) in acetone, rinsed in acetone and dried with filtered air. Full length complementary DNAs (cDNAs) for onco-suppressor 53 (p53), Cyclin-dependent kinase 2 (CDK2), SH2 (Src Homology 2) domain of the proto-oncogene tyrosine-protein kinase (Src) and tyrosine-protein phosphatase non-receptor type 11 (PTPN11) were amplified and cloned. Printing mix was prepared with 0.66 μg/μl DNA capture reagent BG-PEG-NH2 for the one-step synthesis of SNAP-tag substrates from esters on labels or surfaces

Langmuir-Blodgett films when used as nanotemplates for crystallization often leads to marked changes in protein stability and structure. Earlier we found that stability of proteins is also correlated with aqueous surroundings in the crystals. Here we study the direct relationships between presence of LB nanotemplates and unique patterns of water molecules surrounding the protein, for four model proteins for which 3D structures are available, and where crystallization conditions for each protein are the same except the presence of LB nanotemplate. Shape of frequency distribution of volumes occupied by water molecules were analyzed. They were found to be different between "classical" samples of different proteins, but surprisingly quite similar for LB samples. Volumes occupied by each water molecule as the function of the distance of the given molecule from the protein surface were studied. Introduction of LB film leads to appearance of water molecules close to protein surface but occupying large volumes. These findings confirm earlier experimental findings on the role of water molecules in determining protein stability and thereby pointing to water as a possible candidate for differences apparent in LB crystal stability against radiation.

Poly(o-methoxyaniline) and poly(o-methylaniline) were synthesized by oxidative polymerization in the presence of multi-walled carbon nanotubes (MWNT) for the fabrication of chloroform processable nanocomposites obtained by embedding MWNT in the polymer matrix without the formation of covalent bonds. The study of pressure–area isotherms highlighted different substituents along the aromatic rings affected the packing grade of macromolecules when spreading on different subphases in relation to the associated sterical hindrance. The presence of MWNT inside the polymer matrix showed to favor a more stretched conformation of macromolecules with a subsequent increment of area/molecule values with respect to the corresponding pure conducting polymers. Furthermore, the sterical hindrance affected the nanocomposite electrochemical properties and conducting polymers containing less hindering substituents along the aromatic rings turned out to be faster electrochemical systems. Less hindering substituents were also able to enhance the conducting properties of nanocomposite materials in association with MWNT.

Homogeneous matrices of calcium oxide (CaO) were prepared by mixing this material with polyethylene glycol (PEG) acting as malleable inert support in order to obtain processable composites. Preliminary tests were carried out to assess the best concentration of CaO in the composite, individuated in the CaO/PEG weight ratio of 1/4. Experimental data highlighted that the composite was able to selectively detect carbon dioxide (CO2) via a nanogravimetric method by performing the experiments inside an atmosphere-controlled chamber filled with CO2. Furthermore, the composite material showed a linear absorption of CO2 as a function of the gas concentration inside the atmosphere-controlled chamber, thus paving the way for the possible use of these matrices for applications in the field of sensor devices for long-term evaluation of accumulated environmental CO2.

Poly(o-methylaniline) (POTO), poly(o-methoxyaniline) (POAS), poly(2,5-dimethylaniline) (PDMA), poly(2,5-dimethoxyaniline) (PDOA), and nanocomposite based on multi-walled carbon nanotubes (MWNTs) and single-walled carbon nanotubes (SWNTs) embedded in these conducting polymers, were synthesized by oxidative polymerization. We used the Langmuir–Schaefer (LS) technique to fabricate films at the air–water interface and performed the doping process on the undoped films by dipping the substrates in 1 M hydrochloric acid (HCl) aqueous solution. We recorded UV–vis spectra for both the undoped and doped forms and calculated the related band gaps by using the Tauc equation. Experimental data showed the substituents affected the final oxidation ratio of the polymer chains and the presence of carbon nanotubes (CNTs) in the medium of reaction changed the properties in relation of the kind and number of substituents along the aromatic ring. The study of UV–vis spectra of the undoped nanocomposites and the calculated band gaps highlighted that the conducting polymer chains simply wrapped up around CNTs with no strong interaction. Both the kind and number of substituents along the aromatic rings strongly affected the protonation process, since their capability of “tuning” the formation of the polaronic state. The presence of CNTs in the polymer matrix showed no appreciable influence in the chemical properties of the doped nanocomposites with respect to the pure conducting polymers.

A new nanostructured composite film based on thiol gold nanoparticles dispersed in polycarbonate and prepared by evaporating a solution of 1-dodecanthiol gold nanoparticles and polycarbonate was developed for applications as optical lenses. Lenses with superior mechanical properties, coloring and UV ray absorption and with the same transparency as the matrix were obtained. The supporting highly transparent polycarbonate matrix and the chloroform solution of thiol gold nanoparticles, 3 nm mean size, was mixed according to a doping protocol employing different concentrations of thiol gold nanoparticles vs. polycarbonate. The presence of nanoparticles in the polymer films was confirmed by the spectrophotometric detection of the characteristic absorbance marker peak at 540–580 nm. The nanostructured films obtained show a better coverage in the UV-vis range (250–450 nm) even at very low doping ratios, of the order of 1:1,000. These results offer a very promising approach towards the development of efficient nanostructured materials for applications to optical lenses.

The grazing-incidence small-angle X-ray scattering technique has been used here with a microfocus beamline (microGISAXS) to study the effect of temperature on the protein reorganization taking place in a Langmuir-Schaefer multilayered enzyme film. The study appears quite reproducible in the two enzymes being utilized, penicillin G acylase and urease. In-plane and out-of-plane cuts are used to account for the changes in the film thickness and distance between structures taking place by the process of heating up to 423 K and cooling to room-temperature. The out-of-plane cut suggests that the structures are getting closer and are becoming more organized owing to the heating affect. Merging of layers is likely to occur during the heating and cooling process, leading to a loss of correlation between the interfaces of the layers and to the establishment of long-range order. The dramatic increase in long-range order in the Langmuir-Blodgett multilayered enzyme films after heating and cooling, made here apparent by grazing-incidence small-angle X-ray scattering using a microbeam, could in the future open the way to avoiding the bottleneck of protein crystallization for protein structure determination.

Proteins represent versatile building blocks for realization of nanostructured materials to be applied in nanobiotechnology. In the present work, the Langmuir–Blodgett technique was utilized to develop nanobiodevices based on protein molecules. Particularly, lipase thin films were fabricated and characterized, with characterization performed in order to optimize the working parameters. As the first step the protein films were studied at the air–water interface and then transferred onto a solid support for further characterization. The films were characterized by different techniques such as UV–Vis spectroscopy, nanogravimetry, atomic force microscopy, and biochemical assays. Catalytic activity of lipase characterized by the maximal reaction rate found to increase over 10 times as a result of inclusion into LB films, while the substrate binding characterized by the Michaelis constant remain unchanged. Catalytic activity per mole of enzyme was found to increase with the increased number of LB layers up to five, and then decrease at 10, while the surface coverage ranged from 70% to 100% from 1 to 10 layers of lipase. This study exploits the possibility to employ LB based protein structures to use in biocatalysis, exemplified by lipase, which is known as an interfacially-activated enzyme, with olive oil as substrate, when lipase should already be in the maximally active state even without a film. We show, however, that it was possible to form even more active lipase nanostructures by the Langmuir–Blodgett technique at the air–water interface, proving that Langmuir-film provides a better catalytic effect in lipase than a mere oil–water boundary.

Chinese Hamster Ovary fibroblasts (CHO-K1) have shown different protein contents when undergoing differentiation by 3′,5′-cyclic adenosine monophosphate (cAMP), which is known to induce reverse transformation (RT) from malignancy to fibroblast-like characteristics. The mass spectrometry (MS) investigation here reported about the behavior of CHO-K1 cells before and after exposure to cAMP reveals a change in the composition of nuclear proteins associated to an inhibition of the protein expression. Possible implications of this finding on the control of cell reverse transformation are discussed. J. Cell. Biochem. 102: 473–482, 2007. © 2007 Wiley-Liss, Inc.

We present a useful approach towards for biomarkers identification in an innovative self-assembling protein microarray based on “Nucleic Acid Programmable Protein Array” (NAPPA) and SNAP tag coupled to E.coli cell free expression system. This approach prove capable to resolve the “background” problem associated to the above label free detection system for the identification of proteins and of protein-protein interaction in humans that could become used in clinical practice.

In this communication, we report anodic porous alumina (APA) template induced crystallization. The APA nanotemplate was prepared on the glass substrate for the hen egg white lysozyme (HEWL) crystal growth. The changes in the lysozyme crystals morphology, namely in the a/c axis ratio, were observed in the crystal grown by APA nanotemplate, but not in the crystal obtained with classical hanging drop vapor diffusion method, under the same experimental conditions. The comparison of the diffraction data of the two crystals as well as bioinformatics and data mining approaches and molecular dynamics simulations suggest a possible explanation of the nanotemplate crystallization phenomenon and shed light on the APA-induced nanocrystallography.

Background: Protein-protein interactions play a major role in Cancer Control and their detailed understanding by Label-Free Nanotechnology is essential especially within the framework of a personalized medicine-based approach. Materials and Methods: We implemented an array of label-free nanobiotechnologies, including the Quartz Crystal Microbalance with Dissipation factor monitoring (QCM_D). We used it for the conductometric monitoring of an antiblastic (temozolomide) interacting with genes and proteins, such as MLH1, that represents a biomarker of the rate survival of patients suffering from brain tumors, outcome of chemotherapy and resistance to drug itself. We coupled the Nucleic Acid Programmable Protein Arrays (NAPPA) and the cell-free protein array with the quartz crystal microbalance technology. In another proof of principle, we coupled the NAPPA with the SNAP tag E. coli cell-free expression system. The goal is to analyze the protein-protein interaction using Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Bruker Ultraflex and “Protein synthesis Using Recombinant Elements” (PURE) system, thus avoiding the “black box” nature of the cell extract. The E. coli in vitro transcription/translation system (IVTT) in respect to the reticulocyte lysate (RRL) or human lysate (HL) is totally characterized and represents an advantage for the subsequent mass spectrometry (MS) analysis. An R Script for Mass Spectrometry Data Preprocessing before Data Mining (SpADS) provides the user with peak recognition and amplitude independent subtraction functions. The MS samples are obtained from SNAPNAPPA spots and printed on gold coated glass slides in higher density, in order to obtain an amount of protein appropriate for MS analysis. Conclusion: We developed a coherent approach that overcome the drawbacks and pitfalls of the traditional laborious and time-consuming labeled and fluorescencebased experimental procedures. This, taken together with the unique properties of proteins obtained with Langmuir-Blodgett (LB)-based crystallography that can enable new strategies for drug design separately reported, defines our approach to cancer control.

The results of a computer simulation of the lysozyme crystal growth influenced by monomer and tetramer (aggregate) units are discussed. A very recently introduced computer model of biopolymer crystal growth and aggregation is based on the 2D lattice Monte Carlo technique and the coarse-grained HP approximation of the lysozyme monomeric unit. Acceleration of the lysozyme crystal growth by a factor of 4/3, based on the 2AUB (PDB ID) lysozyme unit, obtained from the Langmuir–Blodgett nanotemplate method, has clearly been confirmed by means of the proposed computer simulation. It is concluded that the aggregates (tetramers) involving 2AUB lysozyme crystal growth can be expected to be slightly accelerated when compared to its monomer-based (PDB ID: 193L) counterpart, which is in excellent accord with very recent experimental findings of the emerging applied science called protein nanocrystallography.

Calculation and combined visualization of electrostatic and hydrophobic properties of Cytochrome P450scc based on two very different homology models allowed to identify extensive hydrophobic patches with neutral electrostatic potential and mutations removing such patches and thus expecting to facilitate crystallization of Cytochrome P450scc, especially for the nanotemplate crystallization method. Implications are discussed for optimizing crystallization and other aspects of protein surface properties and protein recognition.

This paper describes a nanoporous substrate for applications in biomedical diagnostics. A photolithographic microstructuring technique for an ordered nanopore array fabrication is reported. The process uses a negative resist with hydrophobic properties increasing specificity to biomolecule linking. Nanoporous alumina is formed by an anodic process and yields straight holes with high aspect ratio: its use as substrates for DNA-microarray or protein-chip application offers several advantages over conventional supports, making them very attractive to use as supports for biological sample microarray application.

Nanocomposite materials of poly(o-anisidine) (POAS) containing titanium dioxide nanoparticles (TiO2), carbon black (CB) and multi-walled carbon nanotubes (MWNT) were prepared. The synthesized materials were deposited in thin films in order to investigate their impedance characteristics by comparison. Electrochemical impedance spectroscopy (EIS) study was performed using the prepared films as working electrodes in a three electrodes configuration cell. Comparison between the fitting parameters was carried out in order to identify the improved nanocomposite for biosensor applications.

A new homology model of bovine cytochrome P450scc is obtained starting from the recently determined crystal structure of mammalian cytochrome P450 2B4. The new emerging structure appears compatible with recent diffraction patterns of bovine P450scc microcrystals as obtained at the Microfocus Beamline of the European Synchrotron Radiation in Grenoble and here reported for the first time. The same atomic structure is utilized thereby to predict the mutations needed for modifying redox potential. A comprehensive comparison is finally carried out with the previous model present in the RCSB Protein DataBank also in terms of the alternative mutations being predicted for the same functional modification. The implication of these studies for optimal sensor construction is discussed.

The present report is dedicated to a systematic comparison of crystal structures produced by the nanobiofilm template method and by the classical hanging-drop vapour-diffusion method. Crystals grown by the innovative nanostructured template method appear indeed radiation-resistant even in the presence of a third-generation highly focused beam at the European Synchrotron Radiation Facility. The implications of this finding for protein crystallography are discussed here in terms of water redistribution and of the detailed atomic resolution comparative studies of the two crystal structures with or without nanobiofilm template, as emerging also from circular-dichroism and thermal denaturation studies.

Chromatin structure, in terms of higher order nuclear-DNA condensation (scanning cytometry) and in terms of acridine orange primary binding sites (flow cytometry), is analyzed and shown to be significantly different between high (B16-F10) and low (B16-F1) metastatic variants of B16 melanoma. Furthermore, double staining of B16-F10 and B16-F1 with ethidium bromide (chromatin) and fluorescamine (membranes) provides the identification of a homogeneous subpopulation of cells with enhanced metastatic potential based on differential fluorescamine uptake. Fluorescamine uptake and poststaining viability is shown to be dependent upon the dye/cell ratio at which staining occurs. Utilizing a sterile cell sorting technique, a subpopulation of B16-F10 with increased fluorescamine uptake representing 30% of the total "intact cell" population was isolated by means of a fluorescence activated cell sorter and replated in vitro. This subpopulation when assayed in vivo produced significantly more pulmonary metastases than its parent cell line. Scanning cytometry of the Feulgen stained sorted subpopulation reveals that the cells possess a unique nuclear morphometry characterized by a 2C-3C DNA content and a large nuclear area (disperse chromatin). Finally, when we assay simultaneously for nuclear-DNA organization and cell membrane organization a progressive uncoupling between nuclear and cell morphometry is apparent if B16-F10 (versus B16-F1).

The interactions between poly{(2,6-pyridinylenevinylene)-co- [(2,5-dioctyloxy-p-phenylene)vinylene]} (PPyPV) and SWNTs have been investigated using UV-Vis absorption spectroscopy. The SWNTs promoted polymer organization. PPyPV is a Lewis base and can be doped by strong and weak Lewis acids. The basicity strength of the PPyPV depended on the polymer interchain interactions, which were enhanced by the presence of SWNTs. As the SWNT concentration was increased, an increment in the K b of PPyPV was observed.

An easy method of incorporating TiO(2) nanoparticles into Nafion perfluorinated ionomer is proposed. Ultrathin films of Nafion were prepared by employing the Langmuir-Schaefer (LS) technique. The pressure-area isotherm study of a Langmuir monolayer of Nafion at the air-water interface on different concentrations of NaCl as the subphase allowed us to find the best experimental conditions for the deposition of stable Langmuir-Schaefer films. Incorporation of TiO(2) nanoparticles was performed by dipping Nafion LS films in a solution of TiO(2) nanoparticles. The uniformity of the TiO(2) incorporation was detected by UV-visible spectroscopy. The morphology of the Nafion, Nafion/TiO(2) nanoparticles thin films, and the changes due to the annealing procedure were investigated by atomic force microscopy. Interestingly, the AFM investigation showed that Nafion and Nafion/TiO(2) LS films have thermal stability up to 600 degrees C.

The lipase-catalyzed degradation of poly(e-caprolactone) (PCL) is described in the present work. The enzymatic degradation of PCL, having number-averaged molecular weight (Mn) of about 87,000 g/mol and molecular weight dispersion (Mw/Mn) of 1.51, was carried out in organic solvent (toluene) and the effects of medium composition, temperature and source of lipase were evaluated and quantified. Three fungal lipases of Candida rugosa, Mucor miehei and Rhizopus delemar were tested to select the best biocatalyst for PCL degradation. The retention of catalytic activity of M. miehei lipase was determined in organic solvent at different temperatures by pH-stat titration, using olive oil as substrate. The experimental data of the activity coefficient versus time were then used to calculate, for each temperature, the corresponding value of the first-order denaturation constant (kd) and to estimate the main thermodynamic parameters of the enzyme thermal denaturation. The results of the degradation reaction, followed by size exclusion chromatography (SEC), showed that M. miehei lipase was able to catalyze the degradation of PCL with a maximum conversion degree of about 70% only after 1 h, within the temperature range of 40–60 °C. The catalytic activity of this biocatalyst demonstrated to be nearly stable at 40 °C, whereas the enzyme half-life was less than 2 h at 60 °C.

A screen-printed enzyme electrode based on flavocytochrome P450scc (RfP450scc) for amperometric determination of cholesterol has been developed. A one-step method for RfP450scc immobilization in the presence of glutaraldehyde or by entrapment of enzyme within a hydrogel of agarose is discussed. The sensitivity of the biosensor based on immobilization procedures of flavocytochrome P450scc by glutaric aldehyde is 13.8 nA microM(-1) and the detection limit is 300 microM with a coefficient of linearity 0.98 for cholesterol in the presence of sodium cholate as detergent. The detection limits and the sensitivity of the agarose-based electrode are 155 microM and 6.9 nA microM(-1) with a linearity coefficient of 0.99. For both types of electrodes, the amperometric response to cholesterol in the presence of detergent was rather quick (1.5-2 min).

A nanocomposite of multiwalled carbon nanotubes (MWNTs) embedded in poly(2,5-dimethylaniline) (PDMA) was synthesised by oxidative polymerisation. The nanocomposite (PDMA–MWNTs) showed a progressive spontaneous undoping process along the time associated to the instability of the doping agent, constituted by HCl, inside the polymeric matrix. The study of the undoping process revealed that this phenomenon is related to the synergetic effect of the sterical hindrance of the substituents on the aromatic rings and the presence of MWNTs inside the polymeric matrix. The conducting properties connected to a doping–undoping equilibrium in the presence of the doping agent were also investigated. The instability of the doping process allowed us to fabricate a spontaneous reversible sensor for acid vapours by setting up a comparative potentiometric circuit and engineering the sensitive element directly on the circuit board.

In the present work the layer-by-layer nano-assembly technique was used for the development of complex catalytic microparticles on the basis of firefly luciferase (FL). FL films containing 1, 2, or 3 monolayers were assembled on silver electrode QCM-resonators and on 520-nm diameter sulfonated polystyrene latex by alternate adsorption of FL and polycations using electrostatic interactions for the interlayer interaction. The assembly process was studied with quartz crystal microbalance, UV-vis spectroscopy, and microelectrophoresis (surface potential). Structural studies of the resulting multilayers confirmed stepwise deposition of FL and cationic poly(dimethyldiallyl ammonium chloride) with a bilayer thickness of 14 nm; a systematic shift of the surface potential from +28 mV for poly(dimethyldiallyl ammonium chloride) to -14 mV for luciferase outermost layer was established. The functionality and stability of the biocolloids were demonstrated by monitoring the intensity of the light emission. Factors influencing the light emitted upon catalytic activity of FL such as the number of luciferase layers in the film and polyion layer at the outermost layer were studied.

Langmuir–Schaefer (LS) films of poly(ortho-anisidine) (POAS) were fabricated by utilizing water and water acidified HCl as subphases, respectively. The uniformity of the films formation and the doping with Nafion were verified by UV–Vis spectroscopy. The morphology and the thickness of the POAS, HCl post-doping POAS and Nafion post-doping POAS LS films were investigated using atomic force microscopy. The electrochemical properties of POAS LS films, HCl post-doping POAS and Nafion post-doping POAS were investigated and compared with our previously published work. The electrochemical switching time of HCl post-doping POAS and Nafion post-doping POAS LS films were also estimated. Keywords: Poly(ortho-anisidine), Nafion, Langmuir–Schaefer, Conducting polymers, Electrochemistry

The recombinant catalytic subunit of human protein kinase CK2 bas been mutagenised at the C-terminal region in an attempt to induce this tail to fold. We suppose in fact that this unstructured C-terminus just might be responsible for the high degradability of the human enzyme. On the basis of theoretical calculations we choose to substitute two distal prolines with alanines (PA 382-384). The mutant bas been purified to the electrophoretic homogeneity by means of three chromatographic steps. By circular dichroism Spectroscopy we verified if the double amino acids substitution reflected on the secondary structure of the recombinant α subunit. According to our theoretical predictions, we observed that the α-helix content of the protein increased when the two distal prolines were substituted by alanines. Moreover the mutant catalytic subunit shows a reduced ability to bind a classical inhibitor such as heparin.

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.

For pt. I see ibid., vol. 1, no. 2, p. 67-72 (2002). A new matrix-integral part of the new DNA microarray instrumentation DNA analyzer (DNASER) is introduced based on a novel DNA patterning on the solid support surface. Such patterning found the way to modify a glass surface for a precise positioning of small droplets of aqueous DNA solutions, without special robots (arrayers), within the boundaries of the modified regions. The physically heterogeneous surface consists of highly hydrophilic spots surrounded by a highly hydrophobic area leading to the surface patterning needed for a DNA microarray: a matrix of hydrophilic spots properly activated for immobilization of oligonucleotides has been fabricated on absolutely passive hydrophobic surface. The optimal efficiency of the above functionalitation technology of a glass-substrate in obtaining DNA microarray was confirmed by the Cy3-dCTP-labeled DNA sample, as shown by charge-coupled device images of the DNASER previously described.

An in-plane patterning process of aggregated nanoparticle thin layers of different inorganic conducting and semiconducting materials produced in Langmuir−Blodgett precursors is developed using film irradiation with an electron beam. This film treatment performs cross-linking of the precursor organic molecules resulting in the insolubility of the hydrocarbon matrix in organic solvents and in the impossibility of particle aggregation in the exposed regions. After immersion of the film into the organic solvent, the exposed areas remain insulating while conducting and semiconducting layers are formed in the nontreated zones due to particle aggregation. The result of patterning is demonstrated by Brewster angle and fluorescent microscopies. The difference of electric properties of treated and nontreated regions is shown by measuring current−voltage characteristics.

Langmuir–Blodgett (LB) films of tert -butyl-substituted copper azaporphyrines, namely binuclear phthalocyanine (Cu 2 Pc 2 ’) and porphyrazine (CuPaz’), were formed from the floating layers differently structured. Effect of the film structure on sorption of benzene and hexane by the LB films was studied microgravimetrically. The greatest benzene/hexane selectivity was found for the homogeneous film of Cu 2 Pc 2 ’.

Present communication is concerned with the application of monolayer engineering, in particular of ‘protective plate’ technique, for the fabrication of alternate-layer assemblies based on enzyme penicillin G acylase. Several structures are compared with each other. The deposited films are tested to determine the values of enzymatic activity and the level of protein detachment in aqueous solutions. As the result, the deposition procedure is found, which enables to obtain biocatalytic media with enhanced performances. The biocatalyst efficiency is proved by three independent techniques including direct yield determination with HPLC. The advantage of applied method of enzyme immobilization with respect to other techniques is demonstrated.

The recently developed ‘protective plate’ method offers the possibility to include protein layers into a Langmuir–Blodgett (LB) assembly without contact of protein molecules with the air–water interface thus avoiding their denaturation. In the present work, this technique was applied for the deposition of biocatalysts with active layers of penicillin G acylase (PGA), an enzyme widely used for medicine production. Easy selection of LB and adsorbed layers resulted in the creation of appropriate environments for the preservation of PGA functions. Two structures were tested regarding such performances as the enzymatic activity value and the level of PGA detachment in aqueous solutions. It was shown that they satisfy the requirements for biocatalytic applications. The enzymatic activity of PGA monolayer incorporated into the film reached 25–30% of the activity value of the equivalent amount of protein in the solution, which is a good result for an immobilized enzyme. Further modification of the deposition procedure resulted in increasing the effective activity per unit of the substrate surface due to adsorption of a thicker protein layer in one cycle. Probably, a three-dimensional frame-like structure was formed, which allowed the substrate molecules to penetrate into the film. The enzymatic activity of such films per unit of the substrate surface was 20–25 times higher than that of the assemblies with one adsorbed monolayer. Finally, the method is proposed of biocatalytic LB assembly deposition onto flexible supports of practically unlimited length without the exposure of protein layer to air medium.

Glutathione S-transferase (GST) fusion cytochrome P4502B4 enzyme obtained by genetic engineering was used in order to optimize the immobilization of the proteins on solid supports. Langmuir–Schaefer, ‘layer-by-layer (LbL)’ and self-assembling techniques were used to form thin films on solid surfaces. In particular, it was studied the possibility to realize alternated structures stabilized by binding affinity between GST-fusion protein and glutathione (GSH) using ‘LbL’ techniques. The characterization of the films was performed by means of π-A isotherms, Brewster angle microscopy and spectrophotometry. Preliminary analysis of the P4502B4 films functionality was realized monitoring the spin-state of the cytochrome P450 by spectrophotometric measurements.

Ultrathin films of poly(thiophene-3-acetic acid) (PTAA)/TiO2 nanocomposite were fabricated layer by layer at various solid surfaces by consecutively adsorbing PTAA and TiCl4 (in toluene–ethanol). The fabrication process was carried out by performing the controlled hydrolysis and condensation processes in separate steps. The build-up of such multilayer films was monitored by UV–VIS spectroscopy and quartz crystal microbalance measurements. The ordered multilayers were subsequently characterized by IR spectroscopy, atomic force microscopy, electrochemical, photo-electrochemical and electrical conductivity measurements. The potential application of such nanocomposite films in photovoltaic applications is discussed.

In order to overcome the difficulties and hurdles too much often encountered in crystallizing a protein with the conventional techniques, our group has introduced the innovative Langmuir-Blodgett (LB)-based crystallization, as a major advance in the field of both structural and functional proteomics, thus pioneering the emerging field of the so-called nanocrystallography or nanobiocrystallography. This approach uniquely combines protein crystallography and nanotechnologies within an integrated, coherent framework that allows one to obtain highly stable protein crystals and to fully characterize them at a nano- and subnanoscale. A variety of experimental techniques and theoretical/semi-theoretical approaches, ranging from atomic force microscopy, circular dichroism, Raman spectroscopy and other spectroscopic methods, microbeam grazing-incidence small-angle X-ray scattering to in silico simulations, bioinformatics, and molecular dynamics, has been exploited in order to study the LB-films and to investigate the kinetics and the main features of LB-grown crystals. When compared to classical hanging-drop crystallization, LB technique appears strikingly superior and yields results comparable with crystallization in microgravity environments. Therefore, the achievement of LB-based crystallography can have a tremendous impact in the field of industrial and clinical/therapeutic applications, opening new perspectives for personalized medicine. These implications are envisaged and discussed in the present contribution.

In this manuscript, we report some proofs-of-principle and preliminary applications and results regarding the analytical quantification of protein expression of some genes biologically and clinically related to cancer. Experiments have been carried out coupling Nucleic Acid Programmable Protein Array (NAPPA) with a recently improved nanogravimetric apparatus which exploits the quartz crystal microbalance with frequency (QCM_F) and quartz crystal microbalance with dissipation monitoring (QCM_D) technologies. The selected proteins are BRIP1, JUN and ATF2. Clinically relevant implications for cancer are envisaged and discussed, as well as future perspectives and developments.

International audience; Micro-beam Grazing-Incidence Small-Angle X-ray scattering (μ-GISAXS), exploiting both the advantages of elastic X-ray scattering and the highly focused third-generation synchrotron radiation micro-beams, is an advanced scattering technique that enables scientists to unravel the details of crystal growth processes and to investigate large-scale structures in thin films, including nanobiofilms or other different kinds of surfaces, such as surface gradients or confined surfaces. In this study, we analyze semi-quantitatively and we simulate our previously acquired μ-GISAXS experiments of Thaumatin and Lysozyme Langmuir-Blodgett (LB)-film, shedding light on nucleation and crystal growth processes. Here, we show that, during LB-thin film facilitated nucleation, the particle radius of Thaumatin and of Lysozyme crystal increases while the film thickness reduces. Structural re-organization inside and within the LB-thin film are likely to lead to the crystal nucleation and growth. These semi-quantitative findings are in agreement with the model previously hypothesized. New insights and implications for protein nanocrystallography are also discussed.

A new method based on Langmuir–Blodgett (LB) technology is presented for the template stimulation of protein crystal growth. The new approach allows the acceleration of the hen egg white lysozyme (HEWL) crystal growth rate in comparison with such a classical vapour diffusion method as hanging drop. Protein thin films were coated on the cover slide of the common crystallization plates. Lysozyme crystal growth was observed on the LB thin films of HEWL.