Arántzazu Narváez

Reagentless biosensors based on self-deposited redox polyelectrolyte-oxidoreductases architectures

Reagentless fructose and alcohol biosensors have been produced with a versatile enzyme immobilisation technique which mimics natural interactions and flexibility of living systems. The electrode architecture is built up on electrostatic interactions by the sequential adsorption of redox polyelectrolytes and redox enzymes giving rise to the efficient transformation of substrate fluxes into electrocatalytic currents. All investigated multilayer structures were self-deposited on 3-mercapto-1-propanesulfonic acid monolayers self-assembled on gold electrodes. Fructose dehydrogenase, horseradish peroxidase (HRP) and the couple HRP-alcohol oxidase were electrochemically connected with a cationic poly[(vinylpyridine)Os(bpy)2Cl] redox polymer (RP) interface in a layer-by-layer self-deposited architecture. The dependence of the distance on the electrochemical response of this interface was also studied showing a clear decrease in the Faradaic current when the distance to the electrode surface was increased. The sensitivities obtained for each biosensor were 19.3, 58.1 and 10.6 mA M(-1) cm(-1) for fructose, H2O2 and methanol, respectively. The sensitivity values can be easily controlled by a rational deposition and manipulation of the charge in the catalytic layers. The electrostatic assembly of the electrochemical interface and the catalytic layers resulted in integrated biochemical systems in which mass transfer diffusion and heterogeneous catalytic and electron transfer steps are efficiently coupled and can be easily manipulated.

Improved mediated tyrosinase amperometric enzyme electrodes

Abstract Tyrosinase amperometric enzyme electrodes in a carbon paste configuration based on the electroreduction of quinoid enzymatic products have been constructed. The electroreduction was mediated by the inclusion in the carbon paste material of an osmium (4,4′-dimethyl 2,2′-bipyridine) 2 (1,10-phenanthroline-5,6-dione) mediator. The mediated electrodes showed a three-fold increase in current density, almost two orders of magnitude decrease in detection limit, and a more than one order of magnitude increase in lifetime of the sensor under FIA conditions compared with the unmediated electrodes. Furthermore, they showed a clear mass transport limited response, changing the response limiting step compared with unmediated electrodes. These findings suggest that the use of suitable mediators and electrode configurations can improve the characteristics of tyrosinase electrodes.

Electrocatalytic oxidation of NADH at graphite electrodes modified with osmium phenanthrolinedione

We report here a detailed study concerning the electrochemical behavior of Os(4,4′-dimethyl, 2,2′-bipyridine)2(1,10-phenanthroline 5,6-dione) complex, adsorbed on spectrographic graphite, and about its electrocatalytic activity for NADH oxidation. Cyclic voltammetric measurements, performed in aqueous phosphate buffer solutions, at different scan rates and pH values, allowed us: (i) to relate the redox response of the o-quinone ligand (phendione) to that of the Os(II) central ion; (ii) to confirm that, in aqueous solutions, the phendione based redox process globally involves two electrons and two protons; (iii) to estimate the rate constant for the heterogeneous electron transfer corresponding to the phendione redox couple (ks≈20.1 s−1). The second order rate constant for electrocatalytic oxidation of NADH (k1,[NADH]=0=1.9×103 M−1 s−1, at pH 6.1) as well as its pH dependence (from pH 5.5 to 8.1) were evaluated from RDE experiments, using both Koutecky–Levich and Lineweaver–Burk data interpretations.

Microperoxidase-11-mediated reduction of hemoproteins: electrocatalyzed reduction of cytochrome c, myoglobin and hemoglobin and electrocatalytic reduction of nitrate in the presence of cytochrome-dependent nitrate reductase

Microperoxidase-11 (MP-11) assembled onto an Au electrode catalyzes the electroreduction of a series of hemoproteins: cytochrome c (Cyt. c) myoglobin (Mb) and hemoglobin (Hb). The electrical contact between MP-11 and the hemoproteins originates from intermolecular complexes between MP-11 and the different hemoproteins. The MP-11-functionalized electrode catalyzes the reduction of cytochrome-dependent nitrate reductase and mediates the bioelectrocatalyzed reduction of nitrate to nitrite. The systems provide assemblies mimicking inter-protein electron transfer in hemoproteins.

Reagentless amperometric glucose dehydrogenase biosensor based on electrocatalytic oxidation of NADH by osmium phenanthrolinedione mediator

The mediator Os(4,4′-dimethyl, 2,2′-bipyridine)2(1,10-phenanthroline-5,6-dione) was used for the catalytic oxidation and recycling of NADH. The mediator, with a redox potential of almost 0 V versus Ag/AgCl, shows clear electrocatalysis of NADH at 0.1 V versus Ag/AgCl at pH 6.0. Carbon paste electrodes modified with the mediator show a clear electrocatalytic wave reaching limiting current densities at 0.15 V versus Ag/AgCl of the order of 140 µA cm–2 l mmol–1 NADH. Reagentless dehydrogenase carbon paste amperometric electrodes for glucose were developed, mixing the mediator, glucose dehydrogenase and NAD+ in the paste. These electrodes were optimized with respect to amounts of enzyme, mediator and NAD+ and were studied with a variety of electrochemical techniques. The results suggest that the response is limited by the enzymic step of the reduction of NAD+ or the oxidation of the substrate. The glucose electrodes show maximum current densities of >0.5 mA cm–2 and very good operational stability in continuous operation; under dry storage conditions their lifetime exceeded 1 month.

Enzyme-modified nanoparticles using biomimetically synthesized silica.

The entrapment of enzymes within biomimetic silica nanoparticles offers unique and simple immobilization protocols that merge the stability of proteins confined in solid phases with the high loading and reduced diffusion limitations inherent to nano-sized structures. Herein, we report on the biomimetic silica entrapment of chemically derivatized horseradish peroxidase for amperometric sensing applications. Scanning electron microscopy shows evidence of the formation of enzyme-modified nanospheres using poly(ethylenimine) as a template for silicic acid condensation. When these nanospheres are directly deposited on graphite electrodes, chemically modified anionic peroxidase shows direct electron transfer at 0 mV vs Ag|AgCl. Microgravimetric measurements as well as SEM images demonstrate that negatively charged peroxidase is also entrapped when silica precipitates at gold electrodes are modified with a self-assembled monolayer of poly(ethylenimine). Electrostatic interactions may play a crucial role for efficient enzyme entrapment and silica condensation at the PEI template monolayer. The in-situ biomimetically synthesized peroxidase nanospheres are catalytically active, enabling direct bioelectrocatalysis at 0 mV vs Ag|AgCl with long-term stability.

Chapter 10 Non-affinity sensing technology: the exploitation of biocatalytic events for environmental analysis

Publisher Summary This chapter focuses on well-recognized and emerging biocatalytic approaches that can be applied to the screening or monitoring of environmentally relevant chemical species for the assessment of good ecological and chemical status. The chapter discusses classic catalytic approaches and well-characterized enzyme reactions as well as other catalytic events in living systems that elicit useful bioanalytical information through catabolic activities. The exploitation of affinity interactions—in particular, immunoreactions—for environmental applications is presented. The chapter describes biosensors within the environmental area for the selection of specific parameters as well as the evaluation of the new monitoring or screening challenges to preserve the environment. The bioengineering tools that are currently expanding and improving the biocatalytic approach in environmental applications are presented in the chapter.

Catalytic and Affinity Amperometric Biosensors for Phenols, Phosphates, and Atrazine: How Transduction Can Improve Performance

Three cases are presented where the rational design of transduction chemistries has led to improved catalytic and affinity electrochemical biosensors for environmental applications. Firstly, the improvement of the reductive recycling of tyrosinase-produced quinones by means of rational modification of electrode surfaces is demonstrated resulting in two orders of magnitude lowering of detection limits, and more than one order of magnitude improvement of the life time of phenolics sensors. Secondly, a phosphorylase A-phosphoglucomutase-glucose 6-phosphate dehydrogenase biosensor is demonstrated, that based on the use of this three-enzyme cascade and combined with new NADH oxidation mediators makes possible reagentless biosensors for phosphate detection. Thirdly, an immunosensor for atrazine is presented that based on electrochemically “wired” peroxidase-labelled atrazine and its competition for the binding sites of immobilised antibodies, reached µg 1−1 (ppb) detection limits and incubation times of minutes.