ИСТИНА

Prussian Blue is recognized as the most advantageous low-potential transducer for hydrogen peroxide [1]. Due to high catalytic activity and selectivity which were comparative with enzyme biocatalysis, we even denoted Prussian Blue as ‘artificial peroxidase’. Recently we reported Prussian Blue based nanozymes [2] defeating natural peroxidase in term of their catalytic activity. Nanozymes were characterized by true enzymatic properties: (i) enzymatic specificity (an absence of oxidase-like activity) and (ii) an ability to operate in physiological solutions. In contrast to bi-enzyme biosensors we propose the nanozyme-enzyme based ones substituting the enzyme peroxidase with the more active and stable Prussian Blue nanoparticles. The procedure of sensor development involves the simple drop-casting of the nanozyme suspension onto the electrode surface with subsequent drying. The resulted modified electrodes characterized with even higher sensitivity to hydrogen peroxide as compared to conventional Prussian Blue sensors. For immobilization of lactate oxidase the improved protocol with enzyme exposing to water-organic mixtures with the high content of organic solvent proposed earlier has been used [3]. Since lactate oxidase is among the less stable enzymes, further stabilization of the enzyme containing membranes is required for multiused commercial biosensors. For this aim we've investigated the stabilization of the lactate oxidase containing siloxane membranes [4] with Nafion analogue – perfluorosulfonated ionomer (PFSI). The perfluorosulfonated ionomer induce the decrease of sensitivity of the resulting biosensors caused by negatively charged substituent able to shield the enzyme active site. However, it is possible to observe an absolute maximum of sensitivity. At the same point the apparent Michaelis constant of the corresponding biosensors displays the minimum for non-zero PFSI content in the membranes illustrating the highest affinity of the immobilized enzyme to lactate. Operation stability of lactate biosensors has been evaluated as the number of lactate injections (0.2 mM) in flow-injection mode remaining the response at the level of 95% from its initial value. The highest operational stability has been registered for biosensors made from the mixture of 1.5 % γaminopropyltriethoxysilane and 0.2 % Nafion, which corresponds to the extremes in sensitivity and Michaelis constant. Operational stability of the corresponding biosensor in terms of the number of lactate injections remaining 95% activity (81 ± 4 injections) is almost 3 times higher as compared to the biosensor made without perfluorosulfonated ionomer (28 ± 3 injections). At the same time the decrease in sensitivity compared to the biosensors made without PFSI is only 35%. Hence, an addition of 0.2% of PFSI to the membrane-forming mixture provides the significantly improved stability of the resulting siloxane based enzyme containing membranes. Thus improved protocol for lactate oxidase immobilization has been used for elaboration of the nanozyme-enzyme biosensor. The resulting nanozyme-enzyme lactate biosensor displays twice higher sensitivity (> 0.2 A∙M−1∙cm−2) compared to the Prussian Blue film based one [5]. Nanozymes “artificial peroxidase” are expected to find wide use in elaboration of oxidase based biosensors.