Colloids and Surfaces B: Biointerfaces 92 (2012) 42–49 Contents lists available at SciVerse ScienceDirect Colloids and Surfaces B: Biointerfaces j our na l ho me p age: www.elsevier.com/locate/colsurfb Graphite grains studded with silver nanoparticles: Description and application in promoting direct biocatalysis between heme protein and the resulting carbon paste electrode Mohammed ElKaoutit a,∗ , Ahmad H. Naggar b , Ignacio Naranjo-Rodríguez a , José L. Hidalgo-Hidalgo de Cisneros a a Departamento de Química Analítica, Facultad de Ciencias, Universidad de Cádiz, 11510 Puerto Real, Cádiz, Spain b Chemistry Department, Faculty of Science, Al-Azhar University, Assiut Branch, 71524 Assiut, Egypt a r t i c l e i n f o Article history: Received 27 July 2011 Received in revised form 24 October 2011 Accepted 8 November 2011 Available online 20 November 2011 Keywords: Studded graphite Silver nanoparticles CPE Heme proteins Peroxide Nitrite a b s t r a c t The impregnation of graphite grains with silver nanoparticles (AgNPs) is proposed for making a novel carbon paste electrode (CPE). The resulting material promotes direct electron transfer and direct biocatal- ysis of embedded heme protein. It is demonstrated that the impregnation of graphite grains with AgNPs of 16–25 nm, incorporated in a CPE, can promote measurable bio-electrochemical phenomena involv- ing hemoglobin and myoglobin. Unlike other biosensors prepared with simple carbon, those based on carbon grains studded with AgNPs show well-defined and quasi-reversible voltammetric peak with het- erogeneous electron transfer rate k s of approximately 0.037 ± 0.007 and 0.013 ± 0.005 s -1 for hemoglobin and myoglobin, respectively. The embedded proteins also retain their bio-catalytical activity for hydro- gen peroxide and nitrite reduction with linear ranges of 0.5–3000 M and 30–150 M, sensitivities of 73.6 ± 0.6 nA M -1 and 5.72 ± 0.11 nA M -1 , and detection limits close to 0.08 M and 5.80 M, for these two analytes respectively. These results support the viability of this preliminary approach for the development of advanced third-generation biosensors. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Since the discovery of the carbon paste electrode (CPE) by Ralph N. Adams in 1958 and its introduction as an alternative to the dropping mercury electrode, particularly in anodic polarography [1], this type of electrode (and sensors based on it) have under- gone considerable development. The scope for development is due to several advantages, such as low cost, low background current, wide potential window, high sensitivity, and renewable surface [2]. However, because the fabrication process involves simply mix- ing graphite with suitable binders to form a paste, modification of this material has branched out across several lines of innova- tion. Between the more relevant ones are: research into alternative binding components such as Teflon [3], sol–gel [4,5], and ionic liquid [6], modification of the paste with polymers, redox media- tors and recognition elements to construct electrochemical sensors and biosensors [7–9], and the doping of the electrode with differ- ent materials such as clay [10], carbon nanotubes [11] and metal nanoparticles to enhance the analytical performance of the associ- ated electrochemical devices [12–18]. Furthermore, in the field of ∗ Corresponding author. Tel.: +34 956016361; fax: +34 956016460. E-mail address: elkaoutit@uca.es (M. ElKaoutit). enzymatic biosensors, the use of metal nanoparticles has attracted much interest in most recent years, possibly due to the advantages inherent in the properties of nanomaterials particularly for improv- ing direct electron transfer between the co-factors and graphite grains. Nevertheless, despite the volume of literature on carbon paste doped with nanoparticles, and its use as a basis for biosensors, only two main methods are being used for the incorporation of such particles in the material: either physical inclusion in the mixed paste or deposition onto the surface of the electrode, starting from a colloidal solution or metallic salt. However, merely deposition of nanoparticles onto the surface of the electrode does not exploit the characteristics of this material to their best advantage, espe- cially the renewable nature of the bio-surface. Physical inclusion by mixing alone produces a biosensor in which the only way to transfer charge is in all cases through the phenomenon of perco- lation (enzyme-metals, enzyme-graphite and metals-graphite), as illustrated in Fig. 1A. Existing processes also have the major limita- tion that only a very limited concentration of nanoparticles can be incorporated, because the proportion of water/mineral oil must be kept low in order to obtain a good paste.Impregnation of graphite grains with metallic nanoparticles is a novel approach for doping the carbon paste electrode with this nanomaterial. So, advanced sensors and biosensors, in which the nano-sized metal particles are 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.11.023