br Introduction Adenosine triphosphate ATP is an

Introduction Adenosine-5′-triphosphate (ATP) is an organic molecule (nucleoside triphosphate) which consists of adenine, ribose, and three phosphoric ZJ 43 residues. ATP serves as a temporary carrier of energy in all living cells, so it is a common substance in any organism. In the cells, new ATP molecules are synthesized during decomposition of organic matter. Energy stored in ATP is utilized for numerous processes of biosynthesis. ATP is also a source of energy for the function of cell membrane proteins, an important precursor of the second messenger-cyclic adenosine monophosphate, an allosteric regulator of a number of cell processes, etc. [1], [2]. Determination of ATP concentration is promising for estimation of the energetic state of cells and tissues. Also, ATP determination may be useful in medicine for studying the biological processes, in which it is involved, namely, the regulation of muscle contraction and platelet aggregation, maintenance of vascular tone, neurotransmission and regulation of the nervous system [3], [4]. The determination of ATP concentration in human blood is promising for the diagnosis of various diseases [5]. The creation of kinase inhibitors may include an evaluation of the amount of ATP used by kinases in the presence of inhibitors and their absence. Modern standard methods of precise determination of ATP concentration, such as spectrophotometry [6] and liquid chromatography [7], require qualified personnel and sophisticated expensive equipment, need complex pretreatment of samples for analysis [8], [9]. Fluorescent, bio- and chemiluminescent methods are free from the above drawbacks; however, often they do not correspond with the demands of ATP monitoring [10]. Radioisotope methods of ATP analysis are highly accurate, but potentially dangerous [11]. Therefore, at present the development of easy-to-use, accurate, fast, selective and low-cost method for determination of ATP concentration in biotechnology and research is an actual challenge. Today there are several laboratory prototypes of biosensors for ATP determination. They are based on pH-sensitive field effect transistors [12], amperometric glassy carbon electrodes [13], amperometric platinum microelectrodes [14], which are usually coated with the enzymes. A common drawback of these biosensors is quite complex structure of electrodes, which increases their cost and reduces the possibility of mass production. Moreover, often two-enzyme systems are used as biorecognition elements of biosensors, what increases overall complexity of the biosensors. Recently, sensors based on photo detection of ATP binding with different receptor molecules were developed [15], [16], [17]. However, the measurement procedure in these cases is quite complicated. An alternative is an application of conductometric biosensors based on planar transducers. These biosensors are advantageous because of simple structure of transducers, low-cost manufacture and fast measurement procedure [18]. Also they do not need a reference or other additional electrodes, and their response time is quite fast. On the other hand, the conductometric transducers are sensitive to all charged substances, including ATP, which presents significant difficulties in measurement of real biological samples. This is the reason why conductometric biosensors are inferior to amperometric and potentiometric biosensors. To the best of our knowledge, no conductometric biosensor for ATP determination has yet been described.
Materials and methods
Results and discussion
Conclusions A reusable conductometric biosensor for determination of ATP and glucose has been developed. The best immobilization of HEX was obtained as a result of HEX and BSA cross-linking by glutaraldehyde during 30min. The biosensor characteristics depended on the composition of working buffer. 5mM HEPES, pH 7.4, with 3mM magnesium ions was found to be the optimal buffer solution. The investigation of relationship between the biosensor sensitivity to ATP and concentration of glucose showed that the optimum glucose concentration was 0.2mM. Limit of ATP detection was 15µM. The relative standard deviation of 10 consecutive measurements of biosensor responses to glucose and ATP was 10.3%. The biosensor developed remained suitable for daily measurements during at least one week. It can be applied for determination of ATP concentration in pharmaceutical vials or other water samples.