XPS and AFM characterization of the enzyme glucose oxidase immobilized on SiO(2) surfaces

A process to immobilize the enzyme glucose oxidase on SiO2 surfaces for the realization of integrated microbiosensors was developed. The sample characterization was performed by monitoring, step by step, oxide activation, silanization, linker molecule (glutaraldehyde) deposition, and enzyme immobilization by means of XPS, AFM, and contact angle measurements. The control of the environment during the procedure, to prevent silane polymerization, and the use of oxide activation to obtain a uniform enzyme layer are issues of crucial importance. The correct protocol application gives a uniform layer of the linker molecule and the maximum sample surface coverage. This result is fundamental for maximizing the enzyme bonding sites on the sample surface and achieving the maximum surface coverage. Thin SiO2 layers thermally grown on a Si substrate were used. The XPS Si 2p signal of the substrate was monitored during immobilization. Such a signal is not completely shielded by the thin oxide layer and it is fully suppressed after the completion of the whole protocol. A power spectral density analysis on the AFM measurements showed the crucial role of both the oxide activation and the intermediate steps (silanization and linker molecule deposition) to obtain uniform immobilized enzyme coverage. Finally, enzymatic activity measurements confirmed the suitability of the optimized protocol.     

Nitrogen‐Doped Ordered Mesoporous Carbon Supported Bimetallic PtCo Nanoparticles for Upgrading of Biophenolics

Hydrodeoxygenation (HDO) is an attractive route for the upgrading of bio‐oils produced from lignocellulose. Current catalysts require harsh conditions to effect HDO, decreasing the process efficiency in terms of energy and carbon balance. Herein we report a novel and facile method for synthesizing bimetallic PtCo nanoparticle catalysts (ca. 1.5 nm) highly dispersed in the framework of nitrogen‐doped ordered mesoporous carbon (NOMC) for this reaction. We demonstrate that NOMC with either 2D hexagonal (p6m) or 3D cubic (Im m) structure can be easily synthesized by simply adjusting the polymerization temperature. We also demonstrate that PtCo/NOMC (metal loading: Pt 9.90 wt %; Co 3.31 wt %) is a highly effective catalyst for HDO of phenolic compounds and “real‐world” biomass‐derived phenolic streams. In the presence of PtCo/NOMC, full deoxygenation of phenolic compounds and a biomass‐derived phenolic stream is achieved under conditions of low severity.     

Uncertainty evaluation for the quantification of low masses of benzo[a]pyrene: Comparison between the Law of Propagation of Uncertainty and the Monte Carlo method

A proper evaluation of the uncertainty associated to the quantification of micropollutants in the environment, like Polycyclic Aromatic Hydrocarbons (PAHs), is crucial for the reliability of the measurement results. The present work describes a comparison between the uncertainty evaluation carried out according to the GUM uncertainty framework and the Monte Carlo (MC) method. This comparison was carried out starting from real data sets obtained from the quantification of benzo[a]pyrene (BaP), spiked on filters commonly used for airborne particulate matter sampling. BaP was chosen as target analyte as it is listed in the current European legislation as marker of the carcinogenic risk for the whole class of PAHs.     

High-throughput mutational screening for beta-thalassemia by single-nucleotide extension

In this work a high-throughput method based on the single-nucleotide extension (SNE) reaction and multicolour detection in a DNA sequencer was developed to screen for eight mutations in the human beta-globin gene: IVSI.110, cd39, IVSI.1, IVSI.6, IVSII.745, HbC, HbS and cd6. The method has been validated on a large number of samples for the two most common mutations causing beta-thalassemia in the Mediterranean area (IVSI.110 and cd39). The development of a high-throughput, fast and reliable method to assay beta-thalassemia mutations represents a significant improvement in molecular diagnosis of this disease. The multicolour detection and the use of multiple injections further enhances the throughput of mutational screening by the DNA sequencer and facilitates automated genotyping for routine molecular diagnostics.     

The genomisotopic approach: a systematic method to isolate products of orphan biosynthetic gene clusters

With the increasing number of genomes sequenced and available in the public domain, a large number of orphan gene clusters, for which the encoded natural product is unknown, have been identified. These orphan gene clusters represent a tremendous source of novel and possibly bioactive compounds. Here, we describe a "genomisotopic approach," which employs a combination of genomic sequence analysis and isotope-guided fractionation to identify unknown compounds synthesized from orphan gene clusters containing nonribosomal peptide synthetases. Analysis of the Pseudomonas fluorescens Pf-5 genome led to the identification of an orphan gene cluster predicted to code for the biosynthesis of a lipopeptide natural product. Application of the genomisotopic approach to isolate the product of this gene cluster resulted in the discovery of orfamide A, founder of a group of bioactive cyclic lipopeptides.     

Diorganoruthenium complexes incorporating noninnocent [C(6)H(2)(CH(2)ER(2))(2)-3,5](2)(2-) (E = N, P) bis-pincer bridging ligands: synthesis, spectroelectrochemistry, and DFT studies

The dinuclear complex [(tpy)RuII(PCP-PCP)RuII(tpy)]Cl2 (bridging PCP-PCP = 3,3',5,5'-tetrakis(diphenylphosphinomethyl)biphenyl, [C6H2(CH2PPh2)2-3,5]22-) was prepared via a transcyclometalation reaction of the bis-pincer ligand [PC(H)P-PC(H)P] and the Ru(II) precursor [Ru(NCN)(tpy)]Cl (NCN = [C6H3(CH2NMe2)2-2,6]-) followed by a reaction with 2,2':6',2' '-terpyridine (tpy). Electrochemical and spectroscopic properties of [(tpy)RuII(PCP-PCP)RuII(tpy)]Cl2 are compared with those of the closely related [(tpy)RuII(NCN-NCN)RuII(tpy)](PF6)2 (NCN-NCN = [C6H2(CH2NMe2)2-3,5]22-) obtained by two-electron reduction of [(tpy)RuIII(NCN-NCN)RuIII(tpy)](PF6)4. The molecular structure of the latter complex has been determined by single-crystal X-ray structure determination. One-electron reduction of [(tpy)RuIII(NCN-NCN)RuIII(tpy)](PF6)4 and one-electron oxidation of [(tpy)RuII(PCP-PCP)RuII(tpy)]Cl2 yielded the mixed-valence species [(tpy)RuIII(NCN-NCN)RuII(tpy)]3+ and [(tpy)RuIII(PCP-PCP)RuII(tpy)]3+, respectively. The comproportionation equilibrium constants Kc (900 and 748 for [(tpy)RuIII(NCN-NCN)RuIII(tpy)]4+ and [(tpy)RuII(PCP-PCP)RuII(tpy)]2+, respectively) determined from cyclic voltammetric data reveal comparable stability of the [RuIII-RuII] state of both complexes. Spectroelectrochemical measurements and near-infrared (NIR) spectroscopy were employed to further characterize the different redox states with special focus on the mixed-valence species and their NIR bands. Analysis of these bands in the framework of Hush theory indicates that the mixed-valence complexes [(tpy)RuIII(PCP-PCP)RuII(tpy)]3+ and [(tpy)RuIII(NCN-NCN)RuII(tpy)]3+ belong to strongly coupled borderline Class II/Class III and intrinsically coupled Class III systems, respectively. Preliminary DFT calculations suggest that extensive delocalization of the spin density over the metal centers and the bridging ligand exists. TD-DFT calculations then suggested a substantial MLCT character of the NIR electronic transitions. The results obtained in this study point to a decreased metal-metal electronic interaction accommodated by the double-cyclometalated bis-pincer bridge when strong sigma-donor NMe2 groups are replaced by weak sigma-donor, pi-acceptor PPh2 groups.     

Loop and Path Spaces and¶Four-Dimensional BF Theories:¶Connections, Holonomies and Observables

We study the differential geometry of principal G-bundles whose base space is the space of free paths (loops) on a manifold M. In particular we consider connections defined in terms of pairs (A,B), where A is a connection for a fixed principal bundle P(M,G) and B is a 2-form on M. The relevant curvatures, parallel transports and holonomies are computed and their expressions in local coordinates are exhibited. When the 2-form B is given by the curvature of A, then the so-called non-abelian Stokes formula follows. For a generic 2-form B, we distinguish the cases when the parallel transport depends on the whole path of paths and when it depends only on the spanned surface. In particular we discuss generalizations of the non-abelian Stokes formula. We study also the invariance properties of the (trace of the) holonomy under suitable transformation groups acting on the pairs (A,B). In this way we are able to define observables for both topological and non-topological quantum field theories of the BF type. In the non-topological case, the surface terms may be relevant for the understanding of the quark-confinement problem. In the topological case the (perturbative) four-dimensional quantum BF-theory is expected to yield invariants of imbedded (or immersed) surfaces in a 4-manifold M. Received: 28 March 1998 / Accepted: 12 September 1998     

Computation of hydration free energies using a parameterized continuum model: Study of equilibrium geometries and reactive processes in water solution

A parameterized self-consistent reaction field model allowing computation of the total free energy of hydration of organic molecules at the ab initio level is presented. The approach uses electrostatic plus polarization energies calculated with the help of a continuum model. The remaining solvation free energy terms are obtained by a simple formula based on atomic parameters and atomic accessible surface areas (ASAs), which are determined with the ASA analytical algorithm. Analytical derivatives of the atomic surfaces areas have been implemented. The atomic parameters have been obtained by a linear regression fit of the calculated and experimental free energies of solution in water for a set of 35 molecules, leading to a standard deviation of 0.75 kcal/mol. Effects of nonelectrostatic terms on solute geometries, association energies, and activation barriers are illustrated. © 1996 by John Wiley & Sons, Inc.     

On the structure of 3‐acetylamino‐5‐methyl‐1,2,4‐oxadiazole and on the fully degenerate rearrangements (FDR) of its anion: a stimulating comparison between the results of ‘in‐silicon chemistry’ and ‘laboratory chemistry’

An accurate crystal structure determination has provided evidence for a planar conformation for 3‐acetylamino‐5‐methyl‐1,2,4‐oxadiazole ( 5 ), in agreement with quantum‐mechanical calculations in the gas phase. In the crystal, a series of strong intermolecular N7H7^….O9 hydrogen bonds link the amido groups of different molecules, causing the formation of infinite parallel ordered chains. The effect of the DMSO solvent on the energy and charge distribution of compound 5 and on its relevant 5 ^? anion, involved in a fully degenerate rearrangement (FDR), has been deepened by quantum‐mechanical DFT calculations. The calculated energy barrier for the FDR increases in going from in vacuo to DMSO, in agreement with previsions based on the Hughes and Ingold rules concerning the nucleophilic substitution of an anionic reagent (the deprotonated amido group in the side chain) on a neutral substrate (the 1,2,4‐oxadiazole ring). Copyright © 2009 John Wiley & Sons, Ltd.     

Optical sensing in microfluidic lab-on-a-chip by femtosecond-laser-written waveguides

We use direct femtosecond laser writing to integrate optical waveguides into a commercial fused silica capillary electrophoresis chip. High-quality waveguides crossing the microfluidic channels are fabricated and used to optically address, with high spatial selectivity, their content. Fluorescence from the optically excited volume is efficiently collected at a 90° angle by a high numerical aperture fiber, resulting in a highly compact and portable device. To test the platform we performed electrophoresis and detection of a 23-mer oligonucleotide plug. Our approach is quite powerful because it allows the integration of photonic functionalities, by simple post-processing, into commercial LOCs fabricated with standard techniques. Figure Femtosecond laser written waveguides can selectively excite fluorescence in a microfluidic channel of a commercial lab-on-a-chip. A compact scheme for on-chip detection by laser induced fluorescence is applied to capillary electrophoresis of a 23-mer Cy3-labeled oligonucleotide