Full Paper Novel Protocol for Covalent Immobilization of Horseradish Peroxidase on Gold Electrode Surface Abd-Elgawad Radi, a * Xavier Mun ˜ oz-Berbel, b Montserrat Cortina-Puig, c Jean-Louis Marty c a Department of Chemistry, Faculty of Science, Mansoura University, 34517 Dumyat, Egypt b Centre Nacional de Microelectro `nica (IMB-CSIC), Esfera UAB, Campus Univ. Auto ´ noma de Barcelona, 08193 Bellaterra, Barcelona, Spain c BIOMEM, Universite ´ de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France *e-mail: abdradi@yahoo.com Received: October 1, 2008 Accepted: November 30, 2008 Abstract A novel protocol for immobilization of horseradish peroxidase (HRP) onto diazonium functionalized screen-printed gold electrode (SPGE) has been successfully developed. This protocol involved 1) electrochemical reduction of p- nitrophenyl diazonium salts synthesized in situ in acidic aqueous solution to graft a layer of p-nitrophenyl on SPGE, 2) electrochemical reduction of the nitro groups to convert to amines, 3) chemical reaction with nitrous acid to transform the amine to diazonium derivative and 4) chemical coupling of the enzyme with the diazonium group to form a covalent diazo bond. The fabricated biosensor showed the direct electrochemistry of HRP and displayed electrocatalytic activity towards the reduction of hydrogen peroxide (H 2 O 2 ) without any mediator. The biosensor exhibited fast amperometric response to H 2 O 2 . The catalytic current increased with increasing H 2 O 2 concentration from 5 mM to 30 mM and the detection limit of the biosensor was 2 mM. The biosensor exhibited acceptable sensitivity, good reproducibility and long-term stability. Keywords: Diazonium salt, Horseradish peroxidase, Direct electrochemistry, Hydrogen peroxide, Screen-printed gold electrode DOI: 10.1002/elan.200804466 1. Introduction The horseradish peroxidase enzyme (HRP) has been involved in numerous applications such as diagnostic assays [1, 2], nucleic acid analysis [3], biosensors [4], bioremedia- tions [5], polymer synthesis [6] and other biotechnological processes [7]. Many of these applications take advantage of the immobilization of HRP on a solid support. In all of these applications, the method of immobilization on a solid surface is a central issue and a key parameter that govern the overall performance of the enzyme. The methods used for the immobilization of HRP include sol-gel encapsulation [8, 9], polymer entrapment [10, 11], physical adsorption (electrostatic [12, 13] and hydrophobic [14 – 16] adhesion), covalent attachment [17 – 21] and biospecific recognition [22, 23]. Among the large variety of immobilization strategies, chemical attachment leading to the formation of irreversible covalent bonds between amino acids or carbohydrates residues in HRP and reactive groups on the support is one of the most widely investigated [17 – 21]. Diazonium organic salt modified electrodes are a promising alternative to conventional electrode modification schemes. In this approach the electrochemical reduction of an aryl diazonium salt creates an aryl centered radical with concomitant release of nitrogen and formation of an aromatic organic layer covalently bonded to the surface. Advantages of this approach are a highly stable surface, ease of preparation, and the ability to synthesize diazonium salts with a wide range of functional groups [24, 25]. The reduction can occur in aqueous and organic media, and can be either spontaneous [26] or electro-induced process. This technique of derivatization has already been demon- strated for a wide range of conducting materials [27 – 33] and semiconducting surfaces [34 – 35]. Herein, a novel multiple-step approach to fabricate an enzyme biosensor is reported. The biosensor fabrication involved 1) grafting of p-nitrophenyl layer on SPGE by electrochemical reduction of p-nitrophenyl diazonium salts synthesized in situ in acidic aqueous solution, 2) the p- nitrophenyl group was then converted to 4-aminophenyl by electrochemical reduction, 3) the 4-aminophenyl was diazo- tized with nitrous acid to form diazonium derivative and 4) the diazonium groups were reacted with phenolic, imida- zole, or amino side chain of the HRP to form covalent diazo bonds [36]. The sequence of steps is shown in Scheme 1. The fabricated biosensor showed the direct electrochemistry of HRP and displayed electrocatalytic activity towards the reduction of H 2 O 2 without any mediator. It exhibited acceptable operational and storage stability. 696 Electroanalysis 2009, 21, No. 6, 696 – 700  2009 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim