| Authors |
Razumienė, Julija ; Gurevičienė, Vidutė ; Šakinytė-Urbikienė, Ieva ; Galuzinska, Liubov ; Seniuk, Igor ; Kravchenko, Vira ; Lytkin, Dmytro |
| Abstract [eng] |
The aim of the study is to create a rapid and reliable biosensor for determining choline in biological media. Materials and methods. Choline oxidase (EC 1.1.3.17, 17 U/mg) from Arthrobacter globiformis was purchased from Sigma. Hydrogen peroxide (30 %, v/v aqueous solution), choline chloride, and bovine serum albumin (V fraction) (BSA) were obtained from Sigma. Glutaraldehyde 25 % was obtained from Merck KGaA. Semipermeable terylene membrane, 12 µM thickness, 0.4 µM diameter of pore were obtained from the Institute Joint Institute of Nuclear Research. The body fluid samples from mice were obtained from the National University of Pharmacy, Ukraine. The layer consisting of 5 µL of a solution comprising ChOx, BSA, and glutaraldehyde was formed on the inner surface of the ring-fixed semipermeable terylene film (working area Ø 2.4 mm) by creating a membrane. Subsequently, it was maintained at 4 °C for a period of 12 hours. The enzymatic membrane was mechanically affixed to the Pt electrode's surface, forming a biosensor. Chronoamperometric measurements were conducted with a custom-made potentiostat (Vilnius University, Life Sciences Centre, Institute of Biochemistry), utilizing a conventional three-electrode electrochemical cell comprising a platinum auxiliary electrode, a saturated Ag/AgCl reference electrode, and the biosensor as the working electrode. To simulate inflammation, we recreated the model of acute toxic tetrachloromethane hepatitis. Hepatitis is an acute or chronic inflammation of the liver, caused by various factors: intoxication with household substances, poisons, drugs, alcohol, autoimmune, and infectious processes. To simulate inflammation, we recreated the model of acute toxic tetrachloromethane hepatitis by the method of O. V. Stefanov. The study was conducted at the Biomedical Research Laboratory of the Educational and Research Institute of Applied Pharmacy of the National University of Pharmacy. Results. A reagentless amperometric choline biosensor was developed and characterized using the enzyme choline oxidase from Arthrobacter globiformis (ChOx). The biosensor showed rapid response, appropriate stability, and sensitivity to choline when acting in model and in real biologic media. Since choline is a product of esterase-catalyzed reactions, the activity of esterases can be evaluated via choline release. This study revealed the increased concentrations of choline in the samples of the model of acute toxic tetrachloromethane hepatitis compared to control animals. Conclusions. The ChOx based biosensor is a reliable tool for the monitoring of choline in biological media, such as blood serum. The activity of esterases can be evaluated via choline release. Consequently, measured esterase activity by choline-type biosensors could serve as biomarkers for the assessment of hepatitis-type inflammation dynamics. This is also highly relevant for the study of the pharmacological action of drugs with the expected antiinflammatory effect. |
