Morosanova Maria A, Morosanova Elena I
Analytical Chemistry Division, Chemistry Department, Lomonosov Moscow State University, 119234 Moscow, Russia.
Gels. 2023 Mar 18;9(3):240. doi: 10.3390/gels9030240.
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.
显色酶促反应对于各种生物化学活性化合物的测定非常方便。溶胶 - 凝胶膜是生物传感器开发的一个有前景的平台。制备固定化酶的溶胶 - 凝胶膜作为创建光学生物传感器的有效方法值得关注。在本工作中，选择条件在聚苯乙烯分光光度计比色皿内部获得掺杂辣根过氧化物酶（HRP）、蘑菇酪氨酸酶（MT）和香蕉粗提物（BE）的溶胶 - 凝胶膜。提出了两种方法：使用四乙氧基硅烷 - 苯基三乙氧基硅烷（TEOS - PhTEOS）混合物作为前驱体，以及使用硅聚乙二醇（SPG）。在这两种类型的膜中，HRP、MT和BE的酶活性均得以保留。基于对掺杂HRP、MT和BE的溶胶 - 凝胶膜催化的酶促反应的动力学研究，我们发现与封装在SPG膜中相比，封装在TEOS - PhTEOS膜中对酶活性的影响较小。固定化对BE的影响明显小于对MT和HRP的影响。封装在TEOS - PhTEOS膜中的BE的米氏常数与未固定化BE的米氏常数几乎没有差异。所提出的溶胶 - 凝胶膜能够测定0.2 - 3.5 mM范围内的过氧化氢（在TMB存在下含HRP的膜），以及分别在0.5 - 10.0 mM和2.0 - 10.0 mM范围内的咖啡酸（含MT和含BE的膜）。含BE的膜已用于以咖啡酸当量测定咖啡中的总多酚含量；分析结果与使用独立测定方法获得的结果高度一致。这些膜非常稳定，在 +4 °C下可保存2个月且活性无损失，在 +25 °C下可保存2周。
