Elsevier

Electrochimica Acta

Volume 63, 29 February 2012, Pages 37-46
Electrochimica Acta

Glassy carbon electrodes modified with hemin-carbon nanomaterial films for amperometric H2O2 and NO2 detection

https://doi.org/10.1016/j.electacta.2011.12.027Get rights and content

Abstract

In this work a new chemical sensor for the H2O2 and nitrite amperometric detection was assembled, using a glassy carbon (GC) bare electrode modified by two different nanocomposite materials. The nanocomposite films were prepared by casting a functionalised carbon nanofiber (CNF-COOH) and single-walled carbon nanotubes (SWCNT-OH, for comparison) on the glassy carbon electrode surface; then an iron(III) protoporphyrin IX (Fe(III)P) was adsorbed on these modified surfaces. A morphological investigation of the nanocomposite layers was also carried out, using the Scanning Electron Microscopy (SEM). The electrochemical characterization, performed optimising several electro-analytical parameters (such as different medium, pH, temperature, scan rate, and potential window), demonstrated that the direct electrochemistry of the Fe(III)P/Fe(II)P redox couple involves 1e/1H+ process. A kinetic evaluation of the electron-transfer reaction mechanism was also carried out, demonstrating that the heterogeneous electron transfer rate constant resulted higher at CNF/hemin/GC biosensor than that evaluated at SWCNT/hemin/GC modified electrode. Finally, the electrocatalytic activity toward the H2O2 reduction was also demonstrated for both sensors but better results were observed working at CNF/hemin/GC modified electrode, especially in terms of an extended linearity (ranging from 50 to 1000 μM), a lower detection limit (L.O.D. = 3σ) of 2.0 × 10−6 M, a higher sensitivity of 2.2 × 10−3 A M−1 cm−2, a fast response time (9 s), a good reproducibility (RSD% < 1, n = 3) and operational stability. In addition, a nitrite electrocatalytic effect was also demonstrated only at CNF/hemin/GC modified electrode, showing an extended linearity (ranging from 5.0 × 10−3 to 2.5 × 10−1 M), a lower detection limit (L.O.D. = 3σ) of 3.18 × 10−4 M, a higher sensitivity of 1.2 × 10−2 A M−1 cm−2, a fast response time of 10 s, a good reproducibility (RSD% <1, n = 3) and finally a good operational stability.

Introduction

The study of direct electron exchange between redox–proteins and an electrode surface represents an interesting subject of investigation in electrochemistry [1], [2], [3] as the process models important biochemical mechanisms (for metabolism and energy transformation) and is relevant to assembly of new and interesting enzyme-based biosensors [4]. From this perspective, hemin (iron(III) protoporphyrin IX) demonstrates interesting chemical properties [5] and could be able to reproduce the redox activity of several heme-proteins (i.e. cytochrome c, catalases, and peroxidases) [6], [7]; but also the oxygen-carrying properties of some proteins [8] (such as myoglobin and hemoglobin). Moreover, as hemin shows an electrocatalytic mechanism similar to that observed for some enzymes, such as peroxidases [5], or nitrite reductase [9], it offers the possibility to detect several compounds, e.g. H2O2 [5], NO/NO2 [9], [10], O2 [11], [12], superoxides [6], and tryptophan [13], and therefore can provide for interesting analytical applications.

Several methods for the immobilization of hemin and other heme proteins on electrode surfaces have been reported in the literature, where biomembrane films [14], kieselgubr films [15], nano gold colloid particles [16], nanostructured titanium oxide [17] and organic solvents [18] were employed and allowed the observation of direct electrochemistry. A relatively new approach involves the possibility to immobilize the heme-proteins on the electrode surface by entrapment in a polymeric matrix [19] created using surfactants [20] or lipids [20], [21], [22], [23]. Recently, functionalised multi-walled (MWCNT-COOH) and single-walled carbon nanotubes (SWCNT-COOH) have also been used to catalyze the electrochemical reaction of several biomolecules, such as β-NADH [24], dopamine [24], [25], epinephrine [24], [26], hemin [27], hemoglobin [28], [29], myoglobin [30], cytocrome c [31], [32], glucose oxidase [33], [34], [35] and peroxidase [36], [37]. Finally, hemin adsorption on a graphite-based electrode and its entrapment in a Nafion matrix, resulting in a significant electrocatalytic effect toward the H2O2 and NO2 reduction, has been reported [27].

In this work hemin immobilization on GC modified electrodes has been carried out using a different functionalised carbon nanomaterials, CNF-COOH and SWCNT-OH. The choice of carbon nanofibers for this study was dictated by its particular properties, especially the larger surface area and its open structure [38] relative to that of nanotubes, thus resulting in a much larger ratio of surface-active groups-to-volume. The higher density of functional groups on the outer surface of carbon nanofibers also allows the selective immobilization and stabilization of functional biomolecules such as proteins, enzymes, and DNA. Also taking into consideration the high conductivity of carbon nanofibers, this material seemed promising for creating the conditions for direct electrochemistry of heme-proteins. Finally, for the first time to our knowledge, H2O2 and NO2 electrocatalysis is observed at a CNF-COOH/hemin/GC modified electrode and is compared with that observed at SWCNT-OH/hemin-modified GC electrode (without the need to use Nafion membranes or other trapping membrane for proteins or enzymes). These systems showed good results especially in terms of a linear range of concentration, a low detection of limit, a high sensitivity and reproducibility, and finally good operational stability.

Section snippets

Materials

Carbon nanofibers (GFE graphitized fibers) were purchased from Electrovac AG (Austria, Europe). Among all the CNFs commercially available from Electrovac AG, we selected CNFs-GFE for having the highest amount of surface functional groups (the total acidity of these carbon nanofibers was determined by direct titration with NaOH, as reported in literature) [38]. They consequently supported the highest degree of biomolecule immobilization. SWCNTs (Carbolex AP-grade) were acquired from Aldrich

Morphological and structural characterization of CNF-based GC electrode

Table 1 summarizes the physical characteristics of the CNFs and SWCNTs. Fig. 1A shows the typical CNF morphology with an average diameter of 80–150 nm. This image is completely different from that obtained for the hemin coated electrode (Fig. 1B), in which globular structures appear. Moreover, the CNF–hemin composite, used to modify the GC electrode surface (see Fig. 1C) shows a very well defined protein layer which completely surrounded the typical carbon nanofiber structure (homogeneously

Conclusions

In this paper a new functionalised carbon nanomaterial (i.e. carbon nanofibers) has been used to immobilize hemin on the GC electrode surface, using a simple and reproducible procedure (by drop-coating). The best electroanalytical performances observed at CNF/H/GC biosensor were compared with those recorded at SWCNT/H/GC biosensor, demonstrating that the open structure of CNFs combined with its higher surface functional group density were responsible for a beneficial effect on the hemin

Acknowledgement

The authors wish to thank the “Regione Lazio project” for financial support.

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