Claudia Danilowicz

Electrical Communication between Electrodes and Enzymes Mediated by Redox Hydrogels.

Different redox polymers based on poly(allylamine) with covalently attached ferrocene and pyridine groups that coordinate iron and ruthenium complexes were prepared, and hydrogels were obtained by cross-linking them with epichlorohydrin. Charge propagation from the underlying electrode, through the redox polymer and electrical communication with the enzyme FADH(2) of glucose oxidase, was studied by cyclic voltammetry and electrochemical impedance spectroscopy. The effects of electrolyte composition, concentration of enzyme and substrate, and electrode potential are reported. The role of different redox mediators covalently attached to the polymer backbone is discussed in terms of driving force and electrostatic barriers.

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.

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.

Liquid chromatography/electrochemical detection of phenols at a Poly[Ni-(Protoporphyrin IX)] chemically modified electrode

Abstract Poly(Ni-(Protoporphyrin IX)) modified glassy carbon electrodes showed an enhanced response for the oxidation of different phenolic compounds ( p -chlorophenol, p -nitrophenol and phenol). It can also suppress the oxidation of more polar substances such as ascorbic acid and potassium hexacyanoferrate(II). These substantial improvements in the sensitivity and selectivity of liquid chromatography with electrochemical detector (LCEC) for the determination of phenols can be obtained by using electrodes thus coated. The method gives a linear range up to 1.3 μg ml −1 and a detection limit of 13 ng ml −1 for p -nitrophenol. The dynamic behaviour of the detector is evaluated with respect to the solute concentration and flow rate. The surface coating is highly stable under the hydrodynamic conditions prevailing in the flow cell.

Effect of ionic strength on the behavior of amperometric enzyme electrodes mediated by redox hydrogels

The electron-transfer behavior of electroactive hydrogels formed by cross-linking ferrocene poly(allylamine) (Fc-PAA) and glucose oxidase is investigated as a function of electrolyte ionic strength using several techniques. Cyclic voltammetry and electrochemical impedance spectroscopy show that the quantity cD(e)(1/2) increases with electrolyte concentration. Enhancement of enzyme catalysis for the oxidation of glucose mediated by Fc-PAA is also apparent at higher KNO(3) concentration. The electroactive redox center concentration, c, and the diffusion coefficient due to electron hopping in the gel, D(e), are independently measured by chronoamperometry at ultramicroelectrodes. Larger electrolyte ionic strength induces an increase in electroactive redox center concentration while D(e) slightly decreases. These results are rationalized in terms of the electrostatic interactions within the redox gel backbone due to water and ion exchange with the external electrolyte, producing swelling and shrinking of the hydrogel.

Comparison between Three Amperometric Sensors for Phenol Determination in Olive Oil Samples

The analytical performance of two modified electrodes (a tyrosinase biosensor and a glassy carbon electrode (GCE) covered with poly[Ni-(protoporphyrin IX)dimethyl ester]) in comparison with the bare electrode has been evaluated in a flow injection system using standard solutions of oleuropein and olive oil samples. The tyrosinase biosensor appeared to be an appropriate device for the selective determination of phenols, moreover it can be easily incorporated in an on-line system. This method was used to evaluate the stability of the samples.

Amperometric Detection of Peroxides Using Peroxidase and Porphyrin Biomimetic Modified Electrodes

Biosensors designed with crosslinked redox hydrogel and horseradish peroxidase have been evaluated in aqueous and nonaqueous media. Electrodes made of a single layer of active material were prepared with Os(byp)2ClPyCH2NHPoly(allylamine) polymer, (PAA-Os), attached to the enzyme by PEG-400 bifunctional reagent with the Os sites acting as electron wires. The electrodes proved to be stable in the presence of organic solvent considering the time evolution of the redox charge and the catalytic current. However, a reasonable decrease in the enzyme response is apparent while increasing solvent content in solution for detection of hydrogen and lauroyl peroxide. As an alternative biomimetic strategy, electrodes were modified with consecutive layers of PAA-Os and electropolymerized Fe-protoporphyrin IX. The electrochemical detection of peroxides was also possible with this system, proving that the poly[Fe-protoporphyrin IX]/Osmium complex system can mimic the peroxidase activity towards hydrogen and organic peroxides. The replacement of Fe-protoporphyrin by Ni-protoporphyrin IX and PAA-Os by PAA in different experiments demonstrates that the mechanism for peroxide reduction involves both Os and Fe-protoporphyrin IX sites. The enzymatic electrodes were applied to benzoyl peroxide quantitation in a pharmaceutical product. The method validation demonstrates that a simple and rapid FIA detection can be applied.

Probing the mechanical stability of DNA in the presence of monovalent cations.

We examine the interaction between monovalent cations and DNA using several different assays that measure the stability of double-stranded DNA (dsDNA). The thermal melting of dsDNA and the mechanical separation of dsDNA into two single strands both depend on the stability of dsDNA with respect to ssDNA and are sensitive to the interstrand phosphate repulsion. We find that the experimentally measured melting temperatures and unzipping forces are approximately the same for all of the ions considered in this study. Likewise, the force required to transform B-DNA into the overstretched form is also similar for all of the ions. In contrast, for a given ion concentration, the force at which the overstretched state fully relaxes back to the canonical B-DNA form depends on the cation; however, for all cations, the overstretching force decreases with decreasing ion concentration, suggesting that this force is sensitive to screening. We observe a general trend for smaller ions to produce more efficient relaxation. Finally, for a given cation, the relaxation can also depend on the anion.