A Novel Collision-Free Homotopy Path Planning for Planar Robotic Arms.

Achieving the smart motion of any autonomous or semi-autonomous robot requires an efficient algorithm to determine a feasible collision-free path. In this paper, a novel collision-free path homotopy-based path-planning algorithm applied to planar robotic arms is presented. The algorithm utilizes homotopy continuation methods (HCMs) to solve the non-linear algebraic equations system (NAES) that models the robot's workspace. The method was validated with three case studies with robotic arms in different configurations. For the first case, a robot arm with three links must enter a narrow corridor with two obstacles. For the second case, a six-link robot arm with a gripper is required to take an object inside a narrow corridor with two obstacles. For the third case, a twenty-link arm must take an object inside a maze-like environment. These case studies validated, by simulation, the versatility and capacity of the proposed path-planning algorithm. The results show that the CPU time is dozens of milliseconds with a memory consumption less than 4.5 kB for the first two cases. For the third case, the CPU time is around 2.7 s and the memory consumption around 18 kB. Finally, the method's performance was further validated using the industrial robot arm CRS CataLyst-5 by Thermo Electron.

Immobilization of Proteins on Gold Surfaces.

Gold has been a widely used support for protein immobilization in a nonspecific way through electrostatic and hydrophobic interactions. As no tools are available to predict the binding of proteins of biological interest to gold supports-for either nano, micro, or macroscopic sizes-smart, reliable, and reproducible protein immobilization protocols on gold are sought. This chapter will focus on a synthetic strategy which allows for the development of a multiplicity of architectures on gold that may be used for protein immobilization. Because of its simplicity, both from a conceptual and a practical point of view, the strategy demonstrated by this step-by-step synthesis of a functionally self-assembled monolayer (SAM) of thiols on gold is accessible to most laboratories working on enzyme technology, even those with limited organic synthesis facilities.

Transforming the canonical piecewise-linear model into a smooth-piecewise representation.

A smoothed representation (based on natural exponential and logarithmic functions) for the canonical piecewise-linear model, is presented. The result is a completely differentiable formulation that exhibits interesting properties, like preserving the parameters of the original piecewise-linear model in such a way that they can be directly inherited to the smooth model in order to determine their parameters, the capability of controlling not only the smoothness grade, but also the approximation accuracy at specific breakpoint locations, a lower or equal overshooting for high order derivatives in comparison with other approaches, and the additional advantage of being expressed in a reduced mathematical form with only two types of inverse functions (logarithmic and exponential). By numerical simulation examples, this proposal is verified and well-illustrated.

Bioelectrochemical oxidation of water.

The electrolysis of water provides a link between electrical energy and hydrogen, a high energy density fuel and a versatile energy carrier, but the process is very expensive. Indeed, the main challenge is to reduce energy consumption for large-scale applications using efficient renewable catalysts that can be produced at low cost. Here we present for the first time that laccase can catalyze electrooxidation of H2O to molecular oxygen. Native and laboratory-evolved laccases immobilized onto electrodes serve as bioelectrocatalytic systems with low overpotential and a high O2 evolution ratio against H2O2 production during H2O electrolysis. Our results open new research ground on H2O splitting, as they overcome serious practical limitations associated with artificial electrocatalysts currently used for O2 evolution.

Influence of the protein structure surrounding the active site on the catalytic activity of [NiFeSe] hydrogenases.

A combined experimental and theoretical study of the catalytic activity of a [NiFeSe] hydrogenase has been performed by H/D exchange mass spectrometry and molecular dynamics simulations. Hydrogenases are enzymes that catalyze the heterolytic cleavage or production of H2. The [NiFeSe] hydrogenases belong to a subgroup of the [NiFe] enzymes in which a selenocysteine is a ligand of the nickel atom in the active site instead of cysteine. The aim of this research is to determine how much the specific catalytic properties of this hydrogenase are influenced by the replacement of a sulfur by selenium in the coordination of the bimetallic active site versus the changes in the protein structure surrounding the active site. The pH dependence of the D2/H(+) exchange activity and the high isotope effect observed in the Michaelis constant for the dihydrogen substrate and in the single exchange/double exchange ratio suggest that a "cage effect" due to the protein structure surrounding the active site is modulating the enzymatic catalysis. This "cage effect" is supported by molecular dynamics simulations of the diffusion of H2 and D2 from the outside to the inside of the protein, which show different accumulation of these substrates in a cavity next to the active site.

Physicochemical characterization of Acidiphilium sp. biofilms.

The biofilm formation of a strain of the extremophile bacterium Acidiphilium sp., capable of donating electrons directly to electrodes, was studied by different surface characterization techniques. We develop a method that allows the simultaneous study of bacterial biofilms by means of fluorescence microscopy and atomic force microscopy (AFM), in which transparent graphitic flakes deposited on a glass substrate are used as a support for the biofilm. The majority of the cells present on the surface were viable, and the growth of the biofilms over time showed a critical increase of the extracellular polymeric substances (EPS) as well as the formation of nanosized particles inside the biofilm. Also, the presence of Fe in Acidiphilium biofilms was determined by X-ray photoelectron spectroscopy (XPS), whereas surface-enhanced infrared absorption spectroscopy indicated the presence of redox-active proteins.

Electricity generation by microorganisms in the sediment-water interface of an extreme acidic microcosm.

The attachment of microorganisms to electrodes is of great interest for electricity generation in microbial fuel cells (MFC) or other applications in bioelectrochemical systems (BES). In this work, a microcosm of the acidic ecosystem of Río Tinto was built and graphite electrodes were introduced at different points. This allowed the study of electricity generation in the sediment/water interface and the involvement of acidophilic microorganisms as biocatalysts of the anodic and cathodic reactions in a fuel-cell configuration. Current densities and power outputs of up to 3.5 A/m² and 0.3 W/m², respectively, were measured at pH 3. Microbial analyses of the electrode surfaces showed that Acidiphilium spp., which uses organic compounds as electron donors, were the predominant biocatalysts of the anodic reactions, whereas the aerobic iron oxidizers Acidithiobacillus ferrooxidans and Leptospirillum spp. were detected mainly on the cathode surface.

Oriented immobilization of a membrane-bound hydrogenase onto an electrode for direct electron transfer.

The interaction of redox enzymes with electrodes is of great interest for studying the catalytic mechanisms of redox enzymes and for bioelectronic applications. Efficient electron transport between the biocatalysts and the electrodes has achieved more success with soluble enzymes than with membrane enzymes because of the higher structural complexity and instability of the latter proteins. In this work, we report a strategy for immobilizing a membrane-bound enzyme onto gold electrodes with a controlled orientation in its fully active conformation. The immobilized redox enzyme is the Ni-Fe-Se hydrogenase from Desulfovibrio vulgaris Hildenborough, which catalyzes H(2)-oxidation reversibly and is associated with the cytoplasmic membrane by a lipidic tail. Gold surfaces modified with this enzyme and phospholipids have been studied by atomic force microscopy (AFM) and electrochemical methods. The combined study indicates that by a two-step immobilization procedure the hydrogenase can be inserted via its lipidic tail onto a phospholipidic bilayer formed over the gold surface, allowing only mediated electron transfer between the enzyme and electrode. However, a one-step immobilization procedure favors the formation of a hydrogenase monolayer over the gold surface with its lipidic tail inserted into a phospholipid bilayer formed on top of the hydrogenase molecules. This latter method has allowed for the first time efficient electron transfer between a membrane-bound enzyme in its native conformation and an electrode.

Original design of an oxygen-tolerant [NiFe] hydrogenase: major effect of a valine-to-cysteine mutation near the active site.

Hydrogenases are efficient biological catalysts of H(2) oxidation and production. Most of them are inhibited by O(2), and a prerequisite for their use in biotechnological applications under air is to improve their oxygen tolerance. We have previously shown that exchanging the residue at position 74 in the large subunit of the oxygen-sensitive [NiFe] hydrogenase from Desulfovibrio fructosovorans could impact the reaction of the enzyme with O(2) (Dementin, S.; J. Am. Chem. Soc. 2009, 131, 10156-10164; Liebgott, P. P.; Nat. Chem. Biol. 2010, 6, 63-70). This residue, a valine in the wild-type enzyme, located at the bottleneck of the gas channel near the active site, has here been exchanged with a cysteine. A thorough characterization using a combination of kinetic, spectroscopic (EPR, FTIR), and electrochemical studies demonstrates that the V74C mutant has features of the naturally occurring oxygen-tolerant membrane-bound hydrogenases (MBH). The mutant is functional during several minutes under O(2), has impaired H(2)-production activity, and has a weaker affinity for CO than the WT. Upon exposure to O(2), it is converted into the more easily reactivatable inactive form, Ni-B, and this inactive state reactivates about 20 times faster than in the WT enzyme. Control experiments carried out with the V74S and V74N mutants indicate that protonation of the position 74 residue is not the reason the mutants reactivate faster than the WT enzyme. The electrochemical behavior of the V74C mutant toward O(2) is intermediate between that of the WT enzyme from D. fructosovorans and the oxygen-tolerant MBH from Aquifex aeolicus.

Electrochemical growth of Acidithiobacillus ferrooxidans on a graphite electrode for obtaining a biocathode for direct electrocatalytic reduction of oxygen.

An aspect in microbial fuel cell research that is currently of great interest is the development of bacterial cathodes. Bacterial cathodes that catalyze oxygen reduction to water at low pH have the advantage of overcoming the kinetic limitations due to the requirement of 4 protons per molecule reduced. In this work we have studied the performance of a biocathode using as electrocatalyst an acidophile microorganism: Acidithiobacillus ferrooxidans. Growth of the microorganism directly on the electrode took place using an applied voltage of 0 V vs. SCE as the only energy source and without adding redox mediators to the solution. Current densities of up to 5 A m(-2) were measured for O2 reduction in the At. ferrooxidans cathode at pH 2.0 and the electrocatalytic wave was shifted 300 mV to higher potential compared to the control graphite electrodes without the bacterium.

Interaction of the active site of the Ni-Fe-Se hydrogenase from Desulfovibrio vulgaris Hildenborough with carbon monoxide and oxygen inhibitors.

The study of Ni-Fe-Se hydrogenases is interesting from the basic research point of view because their active site is a clear example of how nature regulates the catalytic function of an enzyme by the change of a single residue, in this case a cysteine, which is replaced by a selenocysteine. Most hydrogenases are inhibited by CO and O(2). In this work we studied these inhibition processes for the Ni-Fe-Se hydrogenase from Desulfovibrio vulgaris Hildenborough by combining catalytic activity measurements, followed by mass spectrometry or chronoamperometry, with Fourier transform IR spectroscopy experiments. The results show that the CO inhibitor binds to Ni in both conformations of the active site of this hydrogenase in a way similar to that in standard Ni-Fe hydrogenases, although in one of the CO-inhibited conformations the active site of the Ni-Fe-Se hydrogenase is more protected against the attack by O(2). The inhibition of the Ni-Fe-Se hydrogenase activity by O(2) could be explained by oxidation of the terminal cysteine ligand of the active-site Ni, instead of the direct attack of O(2) on the bridging site between Ni and Fe.

Bioelectrochemical studies of azurin and laccase confined in three-dimensional chips based on gold-modified nano-/microstructured silicon.

Double-sided three-dimensional porous silicon chips, 6 mm x 6 mm, covered with a 40 nm gold (nano)layer, were fabricated from a porous silicon wafer. Scanning electron microscopy along with electrochemical characterisation showed sample conductivity, mechanical stability, and high surface area of the thus fabricated devices, viz. 10 times higher electrochemically active surface area compared to the geometric area. The three-dimensional gold coated silicon chips were further modified with thiol layers, followed by immobilisation of a simple copper-containing redox protein, azurin, or a complex multicopper redox enzyme, laccase. The bioelectrochemical studies showed very high surface concentrations of azurin and laccase, i.e. close to the theoretical monolayer coverage. However, direct electron transfer reactions between the biomolecules and gold surfaces were observed only for a small percentage of the immobilised redox protein and enzyme, respectively. Thus, highly efficient oxygen-bioelectroreduction on laccase-modified 3D thiol-gold-porous silicon chips (as compared to planar laccase-modified gold electrodes, 42 microA/cm(2)vs. 7 microA/cm(2), respectively) was obtained only in the presence of an efficient soluble redox mediator.

Introduction of methionines in the gas channel makes [NiFe] hydrogenase aero-tolerant.

Hydrogenases catalyze the conversion between 2H(+) + 2e(-) and H(2)(1). Most of these enzymes are inhibited by O(2), which represents a major drawback for their use in biotechnological applications. Improving hydrogenase O(2) tolerance is therefore a major contemporary challenge to allow the implementation of a sustainable hydrogen economy. We succeeded in improving O(2) tolerance, which we define here as the ability of the enzyme to resist for several minutes to O(2) exposure, by substituting with methionines small hydrophobic residues strongly conserved in the gas channel. Remarkably, the mutated enzymes remained active in the presence of an O(2) concentration close to that found in aerobic solutions in equilibrium with air, while the wild type enzyme is inhibited in a few seconds. Crystallographic and spectroscopic studies showed that the structure and the chemistry at the active site are not affected by the mutations. Kinetic studies demonstrated that the inactivation is slower and reactivation faster in these mutants. We propose that in addition to restricting O(2) diffusion to the active site of the enzyme, methionine may also interact with bound peroxide and provide an assisted escape route for H(2)O(2) toward the gas channel. These results show for the first time that it is possible to improve O(2)-tolerance of [NiFe] hydrogenases, making possible the development of biohydrogen production systems.

Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant: the connection between the C-terminal plug and the conserved (509)VSG(511) tripeptide.

A mutant laccase from the Ascomycete Myceliophthora thermophila has been submitted to iterative cycles of combinatorial saturation mutagenesis through in vivo overlap extension in Saccharomyces cerevisiae. Over 180,000 clones were explored, among which the S510G mutant revealed a direct interaction between the conserved (509)VSG(511) tripeptide, located in the neighborhood of the T1 site, and the C-terminal plug. The K(m)(O)(2) value of the mutant increased 1.5-fold, and the electron transfer pathway between the reducing substrate and the T1 copper ion was altered, improving the catalytic efficiency towards non-phenolic and phenolic substrates by about 3- and 8-fold. Although the geometry at the T1 site was perturbed by the mutation, paradoxically the laccase redox potential was not significantly altered. Together, the results obtained in this study suggest that the (509)VSG(511) tripeptide may play a hitherto unrecognized role in regulating the traffic of oxygen through the C-terminal plug, the latter blocking access to the T2/T3 copper cluster in the native enzyme.

FTIR spectroelectrochemical characterization of the Ni-Fe-Se hydrogenase from Desulfovibrio vulgaris Hildenborough.

For the first time a complete characterization by infrared spectroscopy of a Ni-Fe-Se hydrogenase in its different redox states is reported. The Ni-Fe-Se hydrogenase was isolated from Desulfovibrio vulgaris Hildenborough. Two different electron paramagnetic resonance silent and air-stable redox states that are not in equilibrium were detected. Upon reduction of these states the catalytically active states Ni-R and Ni-C appear immediately. These states are in redox equilibrium and their formal redox potential has been measured. Putative structural differences between the redox states of the active site of the Ni-Fe-Se hydrogenase are discussed.

Laccase electrode for direct electrocatalytic reduction of O2 to H2O with high-operational stability and resistance to chloride inhibition.

Laccase from Trametes hirsuta basidiomycete has been covalently bound to graphite electrodes electrochemically modified with phenyl derivatives as a way to attach the enzyme molecules with an adequate orientation for direct electron transfer (DET). Current densities up to 0.5mA/cm(2) of electrocatalytic reduction of O(2) to H(2)O were obtained in absence of redox mediators, suggesting preferential orientation of the T1 Cu centre of the laccase towards the electrode. The covalent attachment of the laccase molecules to the functionalized electrodes permitted remarkable operational stability. Moreover, O(2) bioelectroreduction based on DET between the laccase and the electrode was not inhibited by chloride ions, whereas mediated bioelectrocatalysis was. In contrast, fluoride ions inhibited both direct and mediated electron transfers-based bioelectrocatalytic reduction of O(2). Thus, two different modes of laccase inhibition by halides are discussed.

Preferential use of an anode as an electron acceptor by an acidophilic bacterium in the presence of oxygen.

Several anaerobic metal-reducing bacteria have been shown to be able to donate electrons directly to an electrode. This property is of great interest for microbial fuel cell development. To date, microbial fuel cell design requires avoiding O(2) diffusion from the cathodic compartment to the sensitive anodic compartment. Here, we show that Acidiphilium sp. strain 3.2 Sup 5 cells that were isolated from an extreme acidic environment are able to colonize graphite felt electrodes. These bacterial electrodes were able to produce high-density electrocatalytic currents, up to 3 A/m(2) at a poised potential of +0.15 V (compared to the value for the reference standard calomel electrode) in the absence of redox mediators, by oxidizing glucose even at saturating air concentrations and very low pHs.

Synthesis of cobalt ferrite core/metallic shell nanoparticles for the development of a specific PNA/DNA biosensor.

Controlled synthesis of cobalt ferrite superparamagnetic nanoparticles covered with a gold shell has been achieved by an affinity and trap strategy. Magnetic nanoparticles are functionalized with a mixture of amino and thiol groups that facilitate the electrostatic attraction and further chemisorption of gold nanoparticles, respectively. Using these nanoparticles as seeds, a complete coating shell is achieved by gold salt-iterative reduction leading to monodisperse water-soluble gold-covered magnetic nanoparticles, with an average diameter ranging from 21 to 29 nm. These constitute a versatile platform for immobilization of biomolecules via thiol chemistry, which is exemplified by the immobilization of peptide nucleic acid (PNA) oligomers that specifically hybridize with complementary DNA molecules in solution. Hybridation with DNA probes has been measured using Rhodamine 6G fluorescence marker and the detection of a single nucleotide mutation has been achieved. These results suggest the PNA-nanoparticles application as a biosensor for DNA genotyping avoiding commonly time-consuming procedures employed.

Optoelectronic properties of nanostructured ensembles controlled by biomolecular logic systems.

A nanostructured system composed of enzyme-functionalized silica microparticles, ca. 74 microm, and gold-coated magnetic nanoparticles, 18 +/- 3 nm, modified with pH-sensitive organic shells was used to process biochemical signals and transduce the output signal into the changes of the optoelectronic properties of the assembly. The enzymes (glucose oxidase, invertase, esterase) covalently bound to the silica microparticles performed Boolean logic operations AND/OR processing biochemical information received in the form of chemical input signals resulting in changes of the solution pH value. Dissociation state of the organic shells on the gold-coated magnetic nanoparticles was controlled by pH changes generated in situ by the enzyme logic systems. The charge variation on the organic shells upon the reversible protonation/dissociation process resulted in the changes of the gold layer localized surface plasmon resonance energy (LSPR), thus producing optical changes in the system. The proton transfer process allowed the functional coupling of the information processing enzyme systems with the signal transducing gold-coated magnetic nanoparticles providing their cooperative performance. Magnetic properties of the gold-coated magnetic nanoparticles allowed separation of the signal-transducing nanoparticles from the enzyme-modified signal processing silica microparticles. The reversible system operation was achieved by the Reset function, returning the pH value and optical properties of the system to the initial state. This process was biocatalyzed by another immobilized enzyme (urease) activated with a biochemical signal. The studied approach opens the way to novel optical biosensors logically processing multiple biochemical signals and "smart" multisignal responsive materials with logically switchable optical properties.