Talanta 78 (2009) 1023–1028 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta Microbiosensor based on glucose oxidase and hexokinase co-immobilised on platinum microelectrode for selective ATP detection O.O. Soldatkin a , O.M. Schuvailo a , S. Marinesco b , R. Cespuglio c , A.P. Soldatkin a,∗ a Biomolecular Electronics Laboratory, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 150 Zabolotny Str., 03143, Kiev, Ukraine b Inserm U628, Université Claude Bernard Lyon I, Lyon, France c EA4170, Université Claude Bernard Lyon 1, 8 Avenue Rockfeller, 69373, Lyon, France article info Article history: Received 3 October 2008 Received in revised form 30 December 2008 Accepted 9 January 2009 Available online 20 January 2009 Keywords: Co-immobilised glucose oxidase and hexakinase Platinum microelectrode Amperometric microbiosensor ATP-analysis Reproducibility Operational and storage stability abstract ATP determination is of great importance since this compound is involved in a number of vital biological processes. To monitor ATP concentration levels, we have developed a microbiosensor based on cylin- drical platinum microelectrode, covered with a layer of poly-m-phenylendiamine (PPD), and layer of co-immobilised glucose oxidase and hexokinase. Conditions for biosensor measurement of ATP (pH, Mg 2+ and substrates concentration) in vitro and microbiosensor characteristics such as sensitivity, selectivity, reproducibility, storage stability were studied and optimized. Under optimal conditions the microbiosen- sor can measure ATP concentrations down to a 2.5 M detection limit with response time about 15 s. Interferences by electroactive compounds like biogenic amines and their metabolites, ascorbic acid, uric acid and l-cystein are rejected in general by the PPD layer. The microbiosensor developed is insensitive to ATP analogues (or substances with similar structure), such as ADP, AMP, GTP and UTP, too. It can be used for ATP analysis in vitro in the reactions consuming or producing macroergic triphosphate molecules to study kinetics of the process and in drug design concerning development of inhibitors specific to target kinases and others target enzymes. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Adenosine-5 ′ -triphosphate (ATP), a well known macroergic sub- stance in all living organisms, plays a key role in the energy turnover of a cell. Its additional functions (the regulation of muscle contraction and platelet aggregation [1], vascular tone and neu- rotransmission [2]) have been revealed as a result of extensive research in several branches of biology and biomedicine over the last decades. ATP determination is of great importance since this compound is involved in a number of vital biological processes. The variation in ATP concentration can exert strong modulatory effects in cen- tral nervous system of mammalian. ATP can influence transmitter release, synaptic plasticity, neurone–glia interactions, nociception, sleep–wake cycles, respiratory and locomotor rhythms, anxiety, depression, aggression and addiction (see Ref. [3] and correspond- ing references from this paper). Moreover, ATP detection can be efficiently used in food industry as a marker of micro-fungal con- tamination and in drug design at development of inhibitors for specific kinases and others enzymes. ∗ Corresponding author. Tel.: +380 44 2000328; fax: +380 44 5260759. E-mail address: a soldatkin@yahoo.com (A.P. Soldatkin). Consequently, there is an actual demand for ATP assays. ATP concentration is usually analyzed using spectrophotometry [4], liq- uid chromatography [5], fluorescence [6], chemiluminescence [7], bioluminescence [8] methods, potentiometric [9–11] and amper- ometric [12–18] biosensors. Among these techniques, biosensors seem to be the most promising tools owing to their characteristics (Table 1). With the help of ATP-biosensors, different biochemical pro- cesses can be studied and visualised directly. An analysis of the characteristics presented in Table 1 shows that ATP potentiometric biosensors demonstrated rather low sensitivity to ATP and strong dependence on buffer capacity. These disadvantages do not permit to use them for ATP measurement in real biological fluid sam- ples at its micromolar physiological concentrations. Concerning amperometric biosensors, there are two main approaches to their fabrication: application of the bi-enzyme system based on (1) glyc- erol oxidase and glycerol kinase (GO/GK) or glycerol kinase and glycerol-3-phosphate oxidase (GK/G-3-PO), and (2) glucose oxidase and hexokinase (GOD/HK). Both approaches give rather good results regarding sensitivity of the developed microbiosensors, however, measurement selectivity and stability presented in Table 1 are not satisfactory enough (unless shown at all). We preferred the devel- opment of the GOD/HK microbiosensor since in this case the system needs glucose for ATP detection, which exists practically in all bio- logical samples. So, second approach is used in our work to develop 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.01.008