F. Pariente

Mechanistic Studies of the Electrocatalytic Oxidation of NADH and Ascorbate at Glassy Carbon Electrodes Modified with Electrodeposited Films Derived from 3,4-Dihydroxybenzaldehyde.

Studies of the electrocatalytic oxidation of β-nicotinamide adenine dinucleotide (NADH) at glassy carbon rotated disk electrodes modified with electrodeposited films derived from 3,4-dihydroxybenzaldehyde (3,4-DHB) indicate that the mechanism of such electrooxidation proceeds via the formation of an intermediate complex. The reaction also appears to be strongly influenced by the presence of Ca(2+) and Mg(2+) ions as well as by pH. Ascorbate can also be electrocatalytically oxidized at these modified electrodes, giving rise to an electrochemical response very similar to that obtained for NADH. Due to this similarity, the presence of ascorbate in NADH determinations presents a severe interference that cannot be mitigated on the basis of electrochemical responses alone. However, this interference effect can be virtually suppressed by the presence of ascorbate oxidase in solution or immobilized on a nylon mesh which, in turn, is in contact with the electrode modified with the film of 3,4-DHB. Using this approach, we describe the construction of an alcohol biosensor based on alcohol dehydrogenase and which is, furthermore, free from interference effects due to ascorbate.

Determination of organophosphorus and carbamic pesticides with an acetylcholinesterase amperometric biosensor using 4-aminophenyl acetate as substrate

Abstract Organophosphorus and carbamic pesticides have been determined with an amperometric acetylcholinesterase-based 4-aminophenyl acetate biosensor. The glassy carbon enzyme membrane covered electrode poised at + 250 mV (vs. sodium chloride saturated calomel electrode) oxidizes the 4-aminophenol formed in the hydrolysis of 4-aminophenyl acetate by acetylcholinesterase in the glutaraldehyde cross-linked layer. The activity of acetylcholinesterase is inhibited in the presence of pesticides. The decrease in activity of the enzyme is monitored by the 4-aminophenyl acetate sensor and is correlated to the concentration of pesticide present in solution. The influence of the acetylcholinesterase loading and the acetylcholinesterase to neutral protein (bovine serum albumin) ratio on the biosensor response was studied and the measuring conditions including pH, substrate concentration, and others were optimized. Detection limits of 4.0 and 13.0 nmol l−1 for paraoxon and carbaryl, respectively, were achieved with a 3-min preincubation time.

Amperometric flow-through biosensor for the determination of pesticides

An amperometric flow-through biosensor for the determination of pesticides is proposed. It is based on the inhibition of the acetyl cholinesterase-catalysed hydrolysis of 4-aminophenylacetate. The calibration graphs were linear from 5.0 X 10 -7 to 1.0X10 -5 M and 5.0X10 -7 to 5.0 X10 -5 M for paroxon and carbaryl, respectively. The detection limit (5% of inhibition) was 1.0 X 10 -7 M pesticide. The relative standard deviations (R.S.D.) (n = 5) were 3.7% for 4.0 X 10 -5 M and 4.0% for 8.0 x 10 -6 M for either carbaryl or paroxon. Electroactive species such as uric and ascorbic acid and benzaldehyde that could be oxidized at the same potential as 4-aminophenol, do not interfere. However, compounds which strongly absorb onto the electrode surface, such as bovine serum albumin (BSA) and surfactants capable of denaturing the enzyme activity, cause an interference. The stability of the sensor was high and even after repetitive use for one month the electrode retained 90% of its original activity. The determination of carbaryl and paroxon was carried out in lagoon water and kiwi fruits. The lowest concentration of pesticide determined in lagoon water was 1.0 X 10 -7 M for paroxon and 4.0 X 10 -7 M for carbaryl.

Amperometric sensor for hypoxanthine and xanthine based on the detection of uric acid

Abstract The construction and response of an immobilized enzyme modified electrode as an amperometric sensor is described. Xanthine oxidase was adsorbed on a carbon paste electrode and physically entrapped with a semipermeable membrane. Uric acid, the product of the enzymatic reaction, was oxidized electrochemically at +0.4 V vs. Ag/AgCl, yielding a steady-state current directly related to the bulk concentration of the substrate. Hypoxanthine and xanthine were determined in the range 5–100 μM at Ph 7.2 with good precision. Interferences are discussed.

Nanostructured rough gold electrodes for the development of lactate oxidase-based biosensors.

The design and characterization of a lactate biosensor using a nanostructured rough gold surface as a transducer is reported. The biosensor is developed by immobilization of lactate oxidase (LOx), on a rough gold electrode modified with a self-assembled monolayer of dithiobis-N-succinimidyl propionate (DTSP). This bifunctional reagent preserves the rough gold structure and allows further covalent immobilization of the enzyme through the terminal succinimidyl groups. The rough gold electrode is characterized using field emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM). The preferential orientation and average crystallite size are obtained by X-ray diffraction (XRD). The resulting lactate oxidase monolayers are characterized by electrochemical impedance spectroscopy (EIS). This nanostructured transducer allows higher mediated electrocatalytic activity than polycrystalline ones. The biosensor response to increasing lactate concentrations, using hydroxymethylferrocene as a redox mediator in solution, is linear up to 1.2 mM with a sensitivity of 1.49 microA mM(-1).

Analytical strategies for amperometric biosensors based on chemically modified electrodes

Various strategies based on the use of chemically modified electrodes for the development of amperometric biosensors are described. Particular emphasis is placed on materials capable of catalyzing the oxidation of NADH and coupling these with enzymatic activities for biosensor construction. In terms of electrocatalysts, the discussion will centre on electrodeposited films of 3,4-dihydroxy benzaldehyde (3,4-DHB) and related analogs as well as on electrodeposited films of transition metal complexes of 1,10-phenanthroline-5,6-dione (phen-dione). Electrodeposited films of these materials have been coupled to the enzymatic activity of aldehyde dehydrogenase and alcohol dehydrogenase for the development of biosensors for aldehydes and ethanol, respectively.

Thermodynamics and kinetics of adsorption and electrocatalysis of NADH oxidation with a self-assembling quinone derivative

Abstract Platinum electrodes have been modified with the imine (I) derived from 3,4-dihydroxybenzaldehyde and 4-aminopyridine; a self-assembling material which incorporates an o -hydroquinone moiety, and a pyridine group. This material adsorbs strongly onto polycrystalline platinum electrodes through the pyridine nitrogen and the resulting modified electrodes retain the redox activity of the quinone/hydroquinone group. The thermodynamics as well as the kinetics of adsorption of this material onto platinum electrodes have been investigated and the apparent diffusion coefficient ( D app ) has also been measured. The modified electrodes exhibit potent and relatively persistent electrocatalysis for NADH oxidation with a diminution of the overpotential of about 300 mV and the electrocatalytic current increases linearly with the NADH concentration from 0.2 mM to 2.0 mM.

Enzyme support systems for biosensor applications based on gold-coated nylon meshes

A novel experimental protocol for enzyme immobilization based on the use of a very permeable support is described and applied to the development of an acetylcholinesterase (AChE) based biosensor. In this system, the enzyme was immobilized onto a gold-coated nylon mesh via a self-assembled monolayer of a bifunctional reagent, cystamine, preadsorbed onto the gold surface. This support has been characterized by optical microscopy and electrochemical measurements of permeability. In the assembled biosensor, the AChE modified mesh was placed over a glassy carbon electrode and the response to 4-aminophenylacetate, used as substrate, was monitored via the enzymatic reaction product, 4-aminophenol, by oxidation at +0.25 V vs. SSCE. This approach to biosensor design has been extended to the determination of organophosphorus and carbamate pesticides by their inhibition of AChE enzymatic activity.

Gold nanoparticles-induced enhancement of the analytical response of an electrochemical biosensor based on an organic-inorganic hybrid composite material

The design and characterization of a new organic-inorganic hybrid composite material for glucose electrochemical sensing are described. This material is based on the entrapment of both gold nanoparticles (AuNPs) and glucose oxidase, which was chosen as a model, into a sol-gel matrix. The addition of spectroscopic grade graphite to this system, which confers conductivity, leads to the development of a material particularly attractive for electrochemical biosensor fabrication. The characterization of the hybrid composite material was performed using atomic force microscopy and scanning electron microscopy techniques. This composite material was applied to the determination of glucose in presence of hydroxymethylferrocene as a redox mediator. The system exhibits a clear electrocatalytic activity towards glucose, allowing its determination at 250 mV vs Ag/AgCl. The performance of the resulting enzyme biosensor was evaluated in terms of sensitivity, detection limit, linear response range, stability and accuracy. Finally, the enhancement of the analytical response of the resulting biosensor induced by the presence of gold nanoparticles was evaluated by comparison with a similar organic-inorganic hybrid composite material without AuNPs.

Influence of macroporous gold support and its functionalization on lactate oxidase-based biosensors response.

A general bioanalytical platform for biosensor applications was developed based on three-dimensional ordered macroporous (3DOM) gold film modified electrodes using lactate oxidase (LOx) as a case study, within the framework of developing approaches of broad applicability. The electrode was electrochemically fabricated with an inverted opal template, making the surface area of the 3DOM gold electrode up to 18 times higher than that of bare flat gold electrodes. These new electrochemical transducers were characterized by using Field Emission Scanning Electron Microscopy (FE-SEM), Atomic Force Microscopy (AFM) and the X-ray diffraction (XRD). The biosensor was developed by immobilization of lactate oxidase (LOx), on a 3DOM gold electrode modified with a self-assembled monolayer of dithiobis-N-succinimidyl propionate (DTSP). The resulting lactate oxidase biosensor was characterized by electrochemical impedance spectroscopy (EIS). The 3DOM gold electrode not only provides a good biocompatible microenvironment but also promotes the increase of conductivity and stability. Thus, the developed lactate oxidase bioanalytical platforms showed higher mediated bioelectrocatalytic activity compared to others previously described based on polycrystalline gold transducers. The response to varying lactate concentrations has been obtained in the presence of hydroxymethylferrocene as redox mediator in solution. Under these conditions, the bioanalytical platform response for DTSP covalently bound enzyme was improved with respect to that obtained in absence of DTSP.