METHOD FOR MEASURING CATECHOLAMINES AND THEIR METABOLITES WITH USING SOLID-PHASE FLUORESCENT BIOSENSOR Russian patent published in 2015 - IPC G01N33/52 

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, and can be applicable for measuring catecholamines and their metabolites in complex array-based objects, including water-soluble, without additional sample preparation. A method is implemented by varying a principal analytical signal shaping and measurement network with the signal recorded in a sensitive biosensor layer; that is followed by a solid-phase fluorescence. The biosensor action is based on enzymatic derivatisation of catecholamines and their metabolites with organic amines (o-phenylene diamine, ethylene diamine) to form quinoxaline derivatives fluorescing in the range of 450-550 nm. The indicator test components are immobilised in the sensitive layer on the biosensor surface; that results in forming and recording the fluorescent signal on the solid surface directly in the reflection mode. The peak fluorescent signal intensity is generated when using a derivatizing agent presented by o-phenyldiamine and performing a process in the horseradish peroxidise concentration of 10-25 nM; in the hydrogen peroxide concentration of 250-500 mcM; in the o-phenylene diamine concentration of 50-100 mcM; in the concentration of catecholamines and their metabolites of 5-2000 nM. Buffer is phosphate buffer 5 mM of pH 9.5-10.0. The sensitive biosensor layer represents a double-layer film {chitosan - o-phenylene diamine/chitosan - peroxidase} applied as a smooth layer on the surface of a glass plate (14×40 mm).

EFFECT: invention provides the simple and sensitive measurement of catecholamines and their metabolites in the objects, which used to be difficult or impossible to analyse using optical detection method for instrumental reasons because the real object has a harmful influence, while the biosensors are insufficiently sensitive and reproducible.

4 cl, 4 dwg