M.S. Gromova, A.V. Kondrashina, A.V. Eremenko, L.V. Sigolaeva, D.V. Volkov, N.I. Zuravleva, I.N. Kurochkin

One major problem of the dairy is mastitis - an inflammatory reaction of the breast. Early diagnosis of mastitis is the most effective way to minimize the damage caused by the disease, therefore, of greatest interest are those clinical diagnostic methods that allow to detect subclinical stages of the disease. One of the important biomarkers of mastitis is NAGase, (N-acetyl-β-D-glucosaminidase (NAGase / NAGaza, EC 3.2.1.52), the enzyme whose level is increased several-fold in the milk at this disease. In this paper we first proposed to use an amperometric biosensor based on tyrosinase obtained by layering of polyelectrolytes technology (CIT) on the surface of graphite electrodes fabricated by screen printing, to determine the activity NAGase. Developed biosensor is based on the measurement of phenol liberated in the hydrolysis of specific substrate phenyl-N-acetyl-β-D-glucosaminide catalyzed by NAGase. Tyrosinase biosensors with a wide linear range (25-10-9 - 1-10-5 M), low detection limit of phenol (12 nM) in buffer solutions, as well as high sensitivity (0,44 A/(M-cm2)) and operational stability (no more than 0.5% for one measurement), allow for the determination of NAGase in the milk of cows. Under optimal conditions, the analysis of the biosensor can detect activity NAGase in whole milk in a wide range 0,8-500 mU/ml, which allows to solve the problem of early diagnosis of mastitis.