Fernando Battaglini

Osmium complexes bearing functional groups: building blocks for integrated chemical systems

Abstract Two new redox-active osmium complexes of the type (Os(bpy)2Cl(pyX))+ have been synthesised, where pyX corresponds to a pyridine derivative bearing a functional group (aldehyde, carboxylate). Their electrochemical behaviour and electronic spectra were studied in acetonitrile and aqueous solutions. The rate coefficients for the re-oxidation of glucose oxidase (GOx) by these new osmium complexes were obtained. The applicability to this system of the equation derived by Nicholson and Shain for an EC′ mechanism is discussed. The mediator containing the carbonyl group was bound to poly(allyl)amine. The new electroactive polymer was wired with oxidases in the construction of glucose and lactate sensors, as an example of the capabilities of these new osmium complexes to build integrated chemical systems.

Layer-by-layer electrostatic deposition of biomolecules on surfaces for molecular recognition, redox mediation and signal generation

Layer-by-layer supramolecular structures composed of alternate layers of negatively charged enzymes and cationic redox polyelectrolyte have been assembled. Glucose oxidase (GOx), lactate oxidase (LOx) and soybean peroxidase (SBP) have been electrically wired to the underlying electrode by means of poly(allylamine) with [Os(bpy)2ClPyCOH]+ covalently attached (PAA-Os) in organized structures with high spatial resolution. Biotinylated glucose oxidase has also been used to assemble step-by-step on antibiotin goat immunoglobulin (IgG) layers and the enzyme was electrically wired by PAA-Os. These spatially organized multilayers with mono- and bienzymatic schemes can work efficiently in molecular recognition, redox mediation and generation of an electrical signal. The concentration of redox mediator integrated into the multilayers, obtained from the voltammetric charge and an estimation of the layer thickness, exceeds by 100-fold the amount of deposited enzyme assessed by quartz crystal microbalance. Differences in GOx electrical wiring efficiency have been detected with the different assembling strategies. The surface concentration of electrically wired enzyme represents a small proportion of all the enzyme molecules present in the multilayers which can be oxidized by the soluble mediator [Os(bpy)2Cl PyCOOH]Cl. This proportion, as well as the rate of FADH2 oxidation by PAA-Os, increases with the number of electrically wired enzyme layers and with the spatial accessibility of the Os moiety to the enzyme active center.

Electrochemical study of sulphonated ferrocenes as redox mediators in enzyme electrodes

Abstract The electrochemical oxidation of mono and disulphonic derivatives of ferrocene (dicyclopentadienyliron) to ferricinium was studied by cyclic voltammetry in aqueous solutions. Both redox systems show fast electrode kinetics on gold, platinum and carbon electrodes. As expected, the sulphonic derivatives are more oxidant than the unsubstituted ferrocene. Ferrocene sulphonates are efficient electron mediators in redox enzyme catalysis for the anaerobic oxidation of glucose catalyzed by glucose oxidase (GOD). The second order rate coefficients for the oxidation of reduced GOD (FADH 2 ), k , are 9.5 × 10 4 and 9.0 ×10 3 1 mol −1 s −1 respectively, for mono and disulphonic derivatives at pH 4.7. Due to the increased solubility of sulphonic derivatives, the complete redox-enzymatic kinetic analysis can be made.

An Os(byp)2ClPyCH2NHPoly(allylamine) hydrogel mediator for enzyme wiring at electrodes

An Os hydrogel based on the covalent attachment of Os(byp)2 ClPyCHO to PAA-NH2 was cross-linked with glucose oxidase (GOx), lactate oxidase (LOx) and horseradish peroxidase (HRP) on GC and Au electrodes by PEG-400 bifunctional reagent. Single layer monoenzyme (GOx or LOx) and bienzyme (HRP-GOx) single layer modified electrodes were prepared with the Os moieties acting as “electron wires or electron shuttles”. Cyclic voltammetry showed diffusional charge propagation in the gel which resulted more stable than similar ferrocene based gels reported before. In solutions containing the substrates, catalytic currents were obtained due to enzyme catalysis for the oxidation of glucose and lactate by the respective enzymes mediated by the Os polymer either by detecting directly the anodic current in a single enzyme electrode or indirectly by further reducing the peroxide formed in the aerobic enzymatic cycle at the Os-wired HRP. A rotating disc electrode (RDE) and a wall jet electrode (WJE) were employed as hydrodynamic electrodes in order to correct the amperometric response for substrate concentration polarization in the external electrolyte.

Electronic tongue for simultaneous detection of endotoxins and other contaminants of microbiological origin.

Endotoxins, also referred to as pyrogens, are lipopolysaccharides (LPS) present in the outer membrane of Gram-negative bacteria, and represent one of the most dangerous microbiological contaminants in water for hemodialysis and intravenous infusion. A method is presented for the simultaneous detection of endotoxins and other bacterial lysis contaminating species in purified water for parenteral formulations. The technique used is electrochemical impedance spectroscopy, with data interpretation using principal component analysis (PCA), cluster analysis (CA), and multivariate discriminant analysis (MDA). Two types of electrode surfaces were modified with LPS recognition agents: (i) a 37 amino acids fragment of a 18 kDa cationic antimicrobial protein (CAP18F) that has LPS binding activity; (ii) the highly selective endotoxin neutralizing protein (ENP). Statistical multivariate analysis of the impedance spectral data allowed the detection of endotoxin at, and below, the threshold pharmaceutical regulatory level. Discrimination of LPS from samples containing proteins, nucleic acids, phospholipids or their mixtures was achieved. These results open a new route to a practical instrumental method capable of detecting and discriminating LPS from other potential pro-inflammatory species of microbiological origin, such as nucleic acids.

Characterization of Lactobacillus Carbohydrate Fermentation Activity Using Immobilized Cell Technique

A microbial bioreactor based on calcium alginate immobilized Lactobacillus cells coupled to a pH electrode was developed for quantitative determination of carbohydrate fermentation activity. A high biomass (1010 cfu mL−1) and particular pregrowth conditions were needed. Reduction of catabolite repression by monosaccharides was achieved by pregrowth in lactose. The evolution of acid production in a continuous flow‐stopped flow bioreactor was monitored for different sugar solutions in contact with the immobilized bacteria. The resulting slopes (ΔmV/Δt) were used to quantify the fermentation capability for a defined sugar related to that of glucose, which was taken as 100%. The procedure is simple, being based on pH variation that can give quantitative results compared to other reported techniques for carbohydrate fermentation pattern from which only qualitative results are obtained. In addition, it offers reduction in time and costs and is a suitable tool for the rapid analysis of isolated strains and in studies of modifications of sugar metabolism in mutants.

Electrochemical Sensing Platform Based on Polyelectrolyte–Surfactant Supramolecular Assemblies Incorporating Carbon Nanotubes

The characterization and application of a polyelectrolyte-surfactant supramolecular assembly formed by poly(allylamine) and dodecyl sulfate (PA-DS) on a screen-printed graphite electrode for the preparation of electrochemical sensing platforms are presented. The system was characterized by X-ray reflectometry (XRR) and grazing-incidence small-angle X-ray scattering (GISAXS) and tested with four benchmark electrochemical probes undergoing different electron-transfer mechanisms on carbon: ferrocyanide, hexaammineruthenium, ascorbic acid, and dopamine. The polyelectrolyte acts as a scaffold favoring the incorporation of the ferrocyanide, an ion oppositely charged to poly(allylamine). Also, its ability to incorporate carbon nanotubes (CNT) is presented. The composite material PA-DS-CNT is able to electrocatalyze the oxidation of dopamine, allowing its detection at micromolar levels in the presence of 100 times higher concentrations of ascorbate and it is shown to be stable, while XRR and GISAXS results confirm a lamellar structure with well-defined domains, not perturbed by the presence of the CNT. The dispersion is easily prepared in aqueous solution and could facilitate the processing of the CNT with an efficient loading and yielding a more robust carbon-based material for sensing applications.

Redox-active concanavalin A: synthesis, characterization, and recognition-driven assembly of interfacial architectures for bioelectronic applications.

The convergence of chemistry, biology, and materials science has paved the way to the emergence of hybrid nanobuilding blocks that incorporate the highly selective recognition properties of biomolecules, with the tailorable functional capabilities of inorganic molecules. In this work, we describe for the first time the decoration of concanavalin A (Con A), a protein with the ability to recognize sugars and form glycoconjugates, with Os(II) redox-active complexes. This strategy enabled the construction of electroactive biosupramolecular materials whose redox potentials could be easily modulated through the facile molecular modification of the electroactive inorganic complexes. Small-angle X-ray scattering (SAXS), steady-state fluorescence, surface plasmon resonance (SPR) spectroscopy, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), and differential-pulsed (DPV) and cyclic voltammetry (CV) were used to characterize the structural and functional features of the synthesized biohybrid building blocks as well as their respective supramolecular assemblies built up on gold electrodes. By harnessing the electroactive and carbohydrate-recognition properties of these tailor-made biohybrid building blocks, we were able to integrate glucose oxidase (GOx) onto gold electrodes via sugar-lectin interactions. The redox activity of the Os-modified Con A interlayer allowed the electronic connection between the multilayered GOx assemblies and the metal electrode as evidenced by the well-defined bioelectrocatalytic response exhibited by the biomolecular assemblies in the presence of the glucose in solution. We consider that this approach based on the spontaneous formation of redox-active biosupramolecular assemblies driven by recognition processes can be of practical relevance for the facile design of biosensors, as well as for the construction of new multifunctional bioelectrochemical systems.

Ascorbate amperometric determination using conducting copolymers from aniline and N-(3-propane sulfonic acid)aniline.

The sequential electrochemical polymerization of aniline and N-(3-propane sulfonic acid)aniline (PSA) is proposed to construct a sensor able to detect ascorbate at physiological conditions. Compared to poly(aniline) modified electrode, a device with improved conducting and electrochemical properties at neutral pH is obtained. The electrochemical copolymerization of the same starting materials is also carried out. For a PSA:aniline ratio of 10:90, a polymer with a similar electrochemical behavior to the one grown in the sequential mode is observed. The detection of ascorbate was tested for both configurations at pH 7.2, the modified electrode is able to determine ascorbate at 0mV versus Ag/AgCl; an optimized sensor constructed by sequential polymerization can easily detect ascorbate concentrations with a detection limit of 2.2muM. Uric acid and dopamine does not interfere in the ascorbate determination.

Recognition-driven layer-by-layer construction of multiprotein assemblies on surfaces: a biomolecular toolkit for building up chemoresponsive bioelectrochemical interfaces

The development of soft bioelectronic interfaces with accurate compositional and topological control of the supramolecular architecture attracts intense interest in the fast-growing field of bioelectronics and biosensing. The present study explores the recognition-driven layer-by-layer assembly of glycoenzymes onto electrode surfaces. The design of the multi-protein interfacial architecture is based on the multivalent supramolecular carbohydrate-lectin interactions between redox glycoproteins and concanavalin A (Con A) derivatives. Specifically, [Os(bpy)(2)Clpy](2+)-tagged Con A (Os-Con A) and native Con A were used to direct the assembly of horseradish peroxidase (HRP) and glucose oxidase (GOx) in a stepwise topologically controlled procedure. In our designed configuration, GOx acts as the biorecognition element to glucose stimulus, while HRP acts as the transducing element. Surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance with dissipation (QCM-D) results are combined to give a close representation of the protein surface coverage and the content of water in the protein assembly. The characterization is complemented with in situ atomic force microscopy (AFM) to give a topographical description of the layers assemblage. Electrochemical (EC) techniques were used to characterize the functional features of the spontaneously self-assembled biohybrid architecture, showing that the whole system presents efficient electron transfer and mass transport processes being able to transform micromolar glucose concentration into electrical information. In this way the combination of the electroactive and nonelectroactive Con A provides an efficient strategy to control the position and composition of the protein layers via recognition-driven processes, which defines its sensitivity toward glucose. Furthermore, the incorporation of dextran as a permeable interlayer able to bind Con A promotes the physical separation of the biochemical and transducing processes, thus enhancing the magnitude of the bioelectrochemical signal. We consider that these results are relevant for the nanoconstruction of functional biointerfaces provided that our experimental evidence reveals the possibility of locally addressing recognition, transduction and amplification elements in interfacial ensembles via LbL recognition-driven processes.