Registration Number
0216U002458,
0113U000142
, R & D reports
Title
Development of new bioanalytical methods of L-, D-lactate and L-arginine analysis for diagnostics of some diseases and control of their treatment
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Head
Gonchar Mykhailo Vasyl'ovych, Кандидат педагогічних наук
Registration Date
16-03-2016
Organization
Institute of cell biology, National Academy of Sciences of Ukraine
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The object of research: bioanalytical important enzymes, biosensor analysis, fluorescent analysis, enzymatic analysis, L-lactate, D-lactate, L-arginine, biological samples. The aim of investigations: A development of new bioanalytical methods for quantitative analysis of some biomarkers important for clinical diagnostics, sports medicine (for example, L- and D-lactate) and control of enzyme therapy (as a model was proposed L-arginine - target for arginine hydrolys by recombinant enzymes - arginase I and arginine deiminase). Methods: microbiological, biochemical, physical, chemical, enzymatic, biosensoric. The results of the work: - two variants of enzyme-fluorescent approaches of the L-arginine analysis - mono enzymatic and bi-enzymatic was developed. The proposed methods are based on: 1) fluorimetric detection of urea - the product of arginase-depended reaction of L-arginine hydrolysis at the interaction with 2,3- butanedione monoxime; 2) fluorescence monitoring the arhinase- and urease-catalyzed hydrolysis product - ammonium ions - the reaction with ortho-phthalic aldehyde. The possibility of using the developed bioanalytical methods for the analysis of L-arginine in the real samples of pharmaceuticals and biological fluids was demonstrated; - a new biosensor method of analysis of L-arginine using recombinant arginine deiminase of bacteria Mycoplasma hominis was developed. The biosensor is based on mono enzyme conversion of L-arginine to citrulline and ammonia with amperometric detection of ammonium ions using chemosensing layer of Nafion-Polyaniline complex; - the preparative isolation and purification of flavocytochrome b2 from the cells of recombinant strain-producer of methylotrophic yeast Ogataea polymorpha (Hansenula polymorpha) was made. The enzyme preparations were immobilized onto different types of nanoparticles and processing protocols of noninvasive enzymatic methods for the analysis of L-lactate in human biological fluids (sweat, saliva) was done; - microbial electrodes based on recombinant yeast cells Ogataea polymorpha tr6 (?CYB2 pAOX-DLD1), capable to overproduction of D-lactate dehydrogenase, cell debris and mitochondria and different electron transfer mediators were constructed; - new prototype of D-lactate-selective sensor, which characterized by high selectivity to D-lactate and insensitivity to interfering influence of accompanying substances in the real samples like ethanol, glucose, pyruvate or L-lactate was developed; - the possibility of using the permeabilized cells of H. polymorpha tr6 and subcellular fractions enriched by mitochondria (as a source of lactate D-dehydrogenase) in photometric analysis of D-lactate based on formation of soluble form of "Berlin blue" (from ferricyanide) and formazan (with nitrotetrazolium blue) was studied; - the model weasel bioreactor based on the cells of producers of L- and D-lactate-specific oxidoreductases immobilized in calcium alginate gel for reception of D-lactate from lactic acid raceme and elimination of toxic D-lactate form model of dairy products was developer and characterised. During 2013-2015 years were published 100 scientific works, including 34 articles (which 23 are International), 7 chapters in English books, 35 manuals, 1 patent of Ukraine for invention, and 55 abstracts.
Product Description
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Іваш М. Ф.
Гайда А. В.
Гайда Г. З.
Демків О. М.
Закальська О. М.
Закальський А.Є.
Куцяба В. І.
Лаврик М. І.
Нечай Г. І.
Прокопів Т. М.
Серкіз Р. Я.
Синенька М. М.
Смуток О. В.
Стасюк Н. Є.
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2020-04-02
