P-Chip and P-Chip Bienzyme Electrodes Based on Recombinant Forms of Horseradish Peroxidase Immobilized on Gold Electrodes

E. E. Ferapontova^1*, V. G. Grigorenko², and A. M. Egorov^2,3

¹Faculty of Pharmacy, Department of Analytical Chemistry, University of Alcala, E-28871 Alcala de Henares, Madrid, Spain; E-mail: elena.ferapontova@uah.es

²Department of Chemical Enzymology, School of Chemistry, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-2742; E-mail: aegorov@enz.chem.msu.ru

³State Scientific Center for Antibiotics, Moscow, Russia; fax: (095) 111-4238; E-mail: egorov@antibiotics.mtu-net.ru

^* To whom correspondence should be addressed.

Received January 26, 2001; Revision received March 5, 2001

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Adsorption and bioelectrocatalytic activity of native horseradish peroxidase (HRP) and its recombinant forms on polycrystalline gold electrodes were studied. Recombinant forms of HRP were produced by a genetic engineering approach using an E. coli expression system. According to direct mass measurements with a quartz crystal microbalance, all the forms of HRP formed monolayer coverage of the enzyme on the gold surface. However, only gold electrodes modified with the recombinant HRP forms (non-glycosylated) exhibited high and stable current response to H₂O₂ due to its bioelectrocatalytic reduction based on direct electron transfer (ET) between gold and the active site of the enzyme. Introduction of a six-His tag either at the C-terminus or at the N-terminus of the enzyme molecule additionally increased the strength of the enzyme binding with the gold surface and the efficiency of direct ET. Immobilization of recombinant forms of HRP containing histidine functional groups on the surface of the gold electrode was used both for the development of a P-chip, a biosensor for hydrogen peroxide determination based on direct ET, and for the development of a bienzyme biosensor electrode for the determination of L-lysine based on co-immobilized recombinant forms of HRP and L-lysine-alpha-oxidase.

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KEY WORDS: recombinant forms of horseradish peroxidase, amperometric biosensor, P-chip, direct electron transfer

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