Abstract
Chromogenic enzymatic reactions are very convenient for the determination of various biochemically active compounds. Sol-gel films are a promising platform for biosensor development. The creation of sol-gel films with immobilized enzymes deserves attention as an effective way to create optical biosensors. In the present work, the conditions are selected to obtain sol-gel films doped with horseradish peroxidase (HRP), mushroom tyrosinase (MT) and crude banana extract (BE), inside the polystyrene spectrophotometric cuvettes. Two procedures are proposed: the use of tetraethoxysilane-phenyltriethoxysilane (TEOS-PhTEOS) mixture as precursor, as well as the use of silicon polyethylene glycol (SPG).In both types of films, the enzymatic activity of HRP, MT, and BE is preserved. Based on the kinetics study of enzymatic reactions catalyzed by sol-gel films doped with HRP, MT, and BE, we found that encapsulation in the TEOS-PhTEOS films affects the enzymatic activity to a lesser extent compared to encapsulation in SPG films. Immobilization affects BE significantly less than MT and HRP. The Michaelis constant for BE encapsulated in TEOS-PhTEOS films almost does not differ from the Michaelis constant for a non-immobilized BE. The proposed sol-gel films allow determining hydrogen peroxide in the range of 0.2-3.5 mM (HRP containing film in the presence of TMB), and caffeic acid in the ranges of 0.5-10.0 mM and 2.0-10.0 mM (MT- and BE-containing films, respectively). BE-containing films have been used to determine the total polyphenol content of coffee in caffeic acid equivalents; the results of the analysis are in good agreement with the results obtained using an independent method of determination. These films are highly stable and can be stored without the loss of activity for 2 months at +4 °C and 2 weeks at +25 °C.