THE FIRST APPLICATION OF SENSORY STRUCTURES BASED ON PHOTOELECTRIC TRANSDUCER FOR THE STUDY OF ENZYMATIC REACTIONS

Abstract

Background. The development of highly sensitive sensor equipment with the possibility of registering analytes in real time is a fast growing research area and a promising diagnostic biomedical technology. Currently, the standard laboratory method for determining the activities of ATPses is an indirect spectroscopic study of the concentration of inorganic phosphate formed as a result of ATP hydrolysis by these enzymes. However, there is no commercially available phosphate sensor with satisfactory parameters of sensitivity, selectivity and stability over time. The purpose of our research was the deve­lopment of a photoelectric recombination sensor system for the real-time detection of biochemical markers and its testing on the example of screening ATPase activity of rat erythrocyte plasma membrane suspension preparations.
Materials and Methods. Experiments were performed on suspension preparations of plasma membranes of erythrocytes of Wistar rats. Preparations of plasma membrane suspensions obtained by Dodge method from each animal were divided into aliquots and used for the simultaneous study of ATPase activity by the reference method of Rathbun & Betlach, as well as the registration of photocurrents induced during the passage of the ATPase reaction using the photoelectric recombination multisensor system of our own design.
Results. The application of silicon sensory structures based on photoelectrical transducer principle for detecting the activity of adenosine triphosphate hydrolases on the example of total Mg²⁺,Na⁺,K⁺-ATPases preparations of plasma membranes of rat erythro­cytes has been experimentally tested. The directly measured analytic parameter is the photocurrent of the deep silicon barrier structure under illumination with high absorption coefficient. The physical features of the device operation have been examined. Detection of such metabolites becomes possible due to reactions intermediates with their own dipole moment (inorganic phosphate, which is one of the products of ATP hydrolysis). The drastic change of photocurrent that characterizes the course of biochemical reaction was observed in real time. The effect is explained by local electrostatic influence on the parameters of recombination centers at the silicon surface that results in surface recombination velocity change. The sensor operation is qualitatively explained in the frame of Stevenson-Keyes’s theory.
Conclusions. Our approach can be regarded as a promising way to elaborate technically simple and highly sensitive method for detection of quantitative behavior of enzymatic reactions. Moreover, the local modification of silicon surface allows obtaining time depending scenarios of the adsorption and thus improving the sensitivity of the sensor. These circumstances open up the possibility of elaborating the complex sensory structures with optimized parameters for certain enzymatic reactions.