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Information × Registration Number 0217U007292, 0116U002208 , R & D reports Title Development of new biosensor and enzymatic methods for determination of the content of creatinine as a biomarker of kidney function and control process of hemodialysis. popup.stage_title Head Gonchar Mykhailo Vasyl'ovych, Кандидат педагогічних наук Registration Date 27-12-2017 Organization Institute of cell biology, National Academy of Sciences of Ukraine popup.description2 The main tasks of the current stage of research were optimization of the preparation of creatinine deiminase (CDI) from recombinant producer's cells, stabilization of the enzyme, its immobilization on microelectrodes and nanocomposite carriers, creation of a laboratory prototype of biosensor selective for creatinine, and elaboration of a new enzymatic method for creatinine assay using recombinant CDI. An optimal scheme for isolation and purification of (His)6-tagged recombinant CDI from the Escherichia coli BL21 transformant cells using metal affinity chromatography on Ni-NTA agarose has been worked out. The stability of the isolated CDI preparations has been studied and various approaches for stabilizing the enzyme have been tested. It was shown that the enzyme is stable over a week at 5 °C in 0.05 M phosphate buffer, pH 7.5 (PB). The enzyme preparations can be stored without significant loss of activity for at least 4 months at -20 ° C in PB with the addition of 20% glycerol or as a suspension in the same buffer at 70% saturation with ammonium sulfate, or in the freeze-dried form in the presence of trehalose as a stabilizer. By the method of electrochemical deposition, hybrid Cu-containing nanoparticles were synthesized on the surface of a graphite electrode. The study of structural, physico-chemical and catalytic properties of the obtained nanoparticles has been carried out and the electrochemical activity of the constructed ammonium-selective chemosensing membrane has been investigated. Electrophoretically homogeneous preparation of CDI with a specific activity of 10 ?mol · min-1 · mg-1 protein was used to create a new amperometric biosensor based on an ammonium-slective chemosensing membrane, prepared by electrochemical deposition of hybrid Cu-containing nanoparticles on the electrode. Different methods of physical and covalent immobilization of CDI on the electrode surface have been tested, and the most optimal approach has been chosen. It includes the formation of a monolayer of ?-mercaptohexadecanoic acid on the surface of Cu-containing nanoparticles, condensation of carboxyl groups with amino groups of the protein by the N-hydroxysuccinimide-carbodiimide method. The designed biosensor is characterized by a wide range of linearity (0.02 - 7 mM), fast response to the analyte (1 min.), satisfactory storage stability (50% preservation of the original signal after 14 days) and a high selectivity to creatinine. Using recombinant CDI, a highly sensitive method for the enzymatic assay of creatinine has been developed, based on spectrophotometric and fluorometric detection of the reaction product (ammonium ions), with a threshold sensitivity of about 6 µM and 0.2 µM, respectively. There were published 19 articles (9 - in international journals, 6 - in Ukrainian journals, 2 chapters in international monograph and 2 - in the Ukrainian book), and 13 abstaracts. 4 articles were submitted for publication in international editions and 1 Ukrainian patent for invention has been preparted. 9 articles are in Scopus. Total Impact Factor of publications is 21,7. Product Description popup.authors Іваш М.Ф. Гайда Г.З. Демків О.М. Закальська О.М. Закальський А.Є. Куцяба В.І. Прокопів Т.М. Серкіз Р.Я. Смуток О.В. Стасюк Н.Є. popup.nrat_date 2020-04-02 Close