EGUÍLAZ RUBIO MARCOS
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Congresos y reuniones científicas
Título:
Development and application of a cytochrome c- modified electrode immobilized on a carbon nanotubes-poly(3-methylthiophene) hybrid composite
Autor/es:
J.M. PINGARRÓN, M. EGUÍLAZ, L. AGÜÍ, P. YÁÑEZ-SEDEÑO
Lugar:
Phoenix
Reunión:
Congreso; 213 th ECS Meeting; 2008
Institución organizadora:
The Electrochemical Society
Resumen:

Preparation of nanostructured electrode surfaces for the design of electrochemical sensing devices has attracted much attention in last years. The resulting electrodes possess advantageous properties derived from the unique characteristics of the nanomaterials used as modifiers. Among these, carbon nanotubes (CNTs) have been extensively employed for the fabrication of electrochemical sensors and biosensors with noticeable features such as an excellent electrocatalytic activity towards the oxidation processes of various molecules, and enhanced sensitivity for the detection of different substances. Recently, efforts have been focused on the design and fabrication of hybrid materials based on CNTs. In this context, CNTs/conducting polymer composites have been used for the preparation of thin film-modified electrodes that exhibit special properties due to the synergic effect from both individual components.

Poly(3-methylthiophene) is an electronically conducting polymer that has been used for the preparation of modified electrodes showing minimization of surface fouling and electrocatalytic effects towards the oxidation of NADH and other compounds. In spite of its advantages and, conversely to other conducting polymers, P3MT has been scarcely applied for the fabrication of composite materials. Recently, we reported a P3MT/CNTs hybrid composite synthesized onto a glassy carbon electrode (GCE). The prepared material showed characteristic properties of both constituents, i.e. good electron transfer ability and large surface area, thus providing excellent electroanalytical responses for the oxidation of NADH and direct electron transfer for cytochrome c and FAD. At the P3MT/CNTs/GCE, cytochrome c showed a quasi-reversible redox pair, with a ∆Ep of 63 mV.

In this communication, cytochrome c (Cyt c) was successfully immobilized onto an L-cysteine-modified P3MT/MWCNTs/GCE. The electrochemical behavior of the resulting Cyt c-modified electrode was studied, and an excellent electrocatalytic ability towards the reduction of H2O2 was observed.

The experimental conditions for the preparation of the Cyt c-modified electrode were optimized. A P3MT/CNTs-GCE was firstly prepared as reported previously (1). Then, this electrode was immersed in a 0.02 M L-Cys solution during 30 min in the darkness. Next, 10 µL of a 0.6 mg ml-1 Cyt c solution prepared in 0.1 M phosphate buffer of pH 7.0 were dropped onto the L-Cys-P3MT/CNTs-GCE surface and the solvent was evaporated at room temperature. During this procedure, the amino acid L-Cys provides negatively charged sites that can interact with positive charges of Cyt c, thus allowing electrostatic immobilization of the metalloprotein (2).

The electrocatalytic behavior of the Cyt c-modified electrode for the reduction of H2O2 was studied by cyclic voltammetry. In the absence of H2O2, the electrode shows a cathodic peak at Ep = -400mV vs Ag/AgCl in 0.05 M Tris-HCl, pH 8.0 solution. When  H2O2 was added to the buffer solution, an increase of the reduction peak current was produced. Amperometric measurements were performed to investigate the dependence of the electrocatalytic current on the H2O2 concentration. A linear range between 0.7 and 400 μM H2O2 was obtained, with a limit of detection of 0.2 μM and a slope value of 1.26 μA M-1. When the H2O2 concentration was higher than 600 μM, a plateau was observed, showing a Michaelis-Menten behavior. The kinetic constant, KMapp, obtained from the Lineweaver-Burk plot, was 451 μM, which is remarkably smaller than those found in the literature (3), this revealing that the Cyt c-L-Cys-P3MT/CNTs-GCE exhibits a high affinity for the substrate.

A good repeatability for the determination of H2O2 was also obtained. A 3.1 % relative standard deviation (RSD) was found for five 1.0 x 10-5 M H2O2 solutions. Moreover, a good electrode-to-electrode reproducibility was observed, with RSD = 8% (n = 5).  The stability was examined by recording the current response towards H2O2 every day. It was found that the electrode retained 85 % of the original response for eight days when it was stored at 4ºC in 0.05 M pH 8.0 PBS solution.

The influence of possible interfering compounds, uric acid, DOPAC, dopamine, catechol, homovanillic acid (HVA), 5-hydroxyindole-3-acetic acid (HIAA), acetaminophen, DMSO and ascorbic acid (AA) was investigated. Only interference was observed for the 1.0 x 10-5 M H2O2 signal when AA was present at the same concentration level. Errors lower than 10 % were found for an AA: H2O2 ratio 0.2:1. This interference can be avoided by casting a Nafion film over the working electrode. Furthermore, the Cyt c-modified hybrid electrode has been applied to the determination of various peroxide species.

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