METHOD OF MEASURING CONCENTRATION OF REACTIVE OXYGEN SPECIES (ROS) IN A SUBCUTANEOUS TUMOR OF LIVING EXPERIMENTAL ANIMALS Russian patent published in 2020 - IPC G01N33/483 

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to experimental medicine, and can be used for in-life observations of the level of reactive oxygen species (ROS) in organs and tissues. Method involves preliminary preparation of experimental animals with subcutaneously grafted tumor, for which animals are anaesthetized by intraperitoneal administration of zoletil solution in concentration 50–75 mg/kg with xylasine in concentration of 5–7.5 mg/kg, providing access to subcutaneous tumor of animal, forming a bath for examination from a skin fold and a subcutaneous back musculature of an animal, filling a bath with a phosphate-salt buffer with pH 7.4, preparing a measuring nanoelectrode, which is a nanopipette filled with pyrolytic carbon with platinum deposited thereon, with preliminary calibration of the nanoelectrode by hydrogen peroxide, placing the experimental animal on the microscope object table, installation of a measuring nanoelectrode in an intravital-electrochemical module (IVEC module) holder, mutual positioning of a tumor and a measuring nanoelectrode, placing the silver chloride reference electrode in a bath with a phosphate-salt buffer solution, connecting it to a measuring system, supplying a linear potential sweep from -800 mV to +800 mV, step-by-step introduction of the nanoelectrode into the tumor at a given depth at a given angle and measuring current strength at potential of +800 mV to determine the level of ROS at each step of immersing the nanoelectrode, determining the value of the ROS concentration in the tumor by the corresponding calibration curve, for construction of which a nanoelectrode and a comparison electrode are connected to instruments for removing current-voltage characteristics and successively lowered into aqueous solutions of hydrogen peroxide with a known concentration in range of 10^-7 to 10^-4 mol/l, potential sweep from -800 mV to +800 mV relative to silver chloride reference electrode is applied to nanoelectrode and current value is measured at +800 mV in each water solution of hydrogen peroxide and calibration curve is plotted, where concentration of hydrogen peroxide is given on one axis, and on other axis there is concentration of hydrogen peroxide, and on other axis – magnitude of current strength.

EFFECT: use of the invention enables to determine concentration of ROS inside a tumor of a living experimental animal at a given point with high spatial and temporal resolution, which enables to obtain a complete picture of ROS distribution in the tumor volume.

4 cl, 1 ex, 6 dwg