The dissertation is devoted to the development of fundamental and technological bases for the creation of multi-enzyme electrochemical biosensors for practical application in various fields of human activity.
An important stage in the creation of any multi-enzyme biosensor is the testing and preparation of electrochemical transducers. Thus, the corresponding methods are developed for amperometric and conductometric transducers. The procedures are proposed to compare the measuring instruments and circuits of analysis. These procedures are mainly the same for amperometry and conductometry, but they can differ in specific experiments.
The first variant of multienzyme biosensors is based on the cascades of enzymatic reactions resulting in gradual transformation of the substrate into an electroactive product, which is registered by electrochemical transducers. In the work, the biosensors developed for the determination of lactose, maltose, arginine and acetylcholine were based on various cascades of enzymatic reactions. At the first stage, the initial biosensor responses and the calibration curves are obtained for the initial variant of device. The next important step in the multibiosensor development is optimization of basic parameters of immobilization, i.e. concentrations of enzymes and crosslinking agent and duration of the process. Then the influence of the parameters of the analyzed solution on the work of the biosensor was studied. It is also necessary to explore such parameters as signal reproducibility, operational stability, storage stability, repeatability of biosensor preparation, selectivity for possible interferents, etc. The last step in the development of biosensors is to test their analytical characteristics, such as sensitivity towards substrate, linear working range, minimal limit of detection, baseline noise and drift, measurement error, etc.
The next task was to evaluate the chances of biosensors based on the competition of different enzymes in the bioselective element for the target substrate and to analyze their peculiarities. Two bienzyme biosensors for determination of ATP and glutamate have been developed.
When creating the bienzyme biosensor for the glutamate determination considerable efforts have been made to improve its selectivity with respect to electroactive substances. It was found when ascorbate oxidase was added to the bioselective element of a biosensor based on glutamate oxidase, the response to glutamate remained constant whereas the response to ascorbic acid significantly reduced.
Two multienzyme biosensors have been developed for indirect determination of the substances. The biosensor for analysis of the creatine kinase activity was created. The second biosensor was based on the effect of inhibition enzyme by heavy metal ions.
Using the created laboratory prototypes of multi-enzyme biosensors, a number of real biological, pharmaceutical, food and environmental samples were analyzed. The biosensor data are verified by traditional methods of analysis and the high level of correlation of the obtained results is shown.
Thus, the scientific and technological bases for creating multienzyme electrochemical biosensors are substantiated and elaborated for practical use in medical diagnostics, environmental protection, agriculture, monitoring biotechnological processes, control the quality of food.
