Gunaratna P C, Wilson G S
Department of Chemistry, University of Kansas, Lawrence 66045.
Anal Chem. 1990 Feb 15;62(4):402-7. doi: 10.1021/ac00203a017.
Immobilized enzyme reactors have been used with high-performance liquid chromatography (HPLC) and electrochemical detection to detect acetylcholine and choline in brain tissue samples. Acetylcholine and choline eluting from the LC column are introduced into a reactor containing immobilized acetylcholinesterase, which hydrolyzes acetylcholine to choline. The product is converted by a second enzyme, choline oxidase, to hydrogen peroxide, which is determined amperometrically. Several novel immobilization techniques including immobilization through enzyme-specific antibodies were used to immobilize these enzymes to retain maximum activity. Improved detection limits were observed when the enzymes were immobilized through the avidin-biotin linkage. Better sensitivity and detection limit were obtained when both enzymes were immobilized together on the same support through the avidin-biotin linkage than when they were separately immobilized and used in two columns. The postcolumn system was applied to brain tissue extracts.
固定化酶反应器已与高效液相色谱（HPLC）和电化学检测联用，用于检测脑组织样本中的乙酰胆碱和胆碱。从液相色谱柱洗脱的乙酰胆碱和胆碱被引入含有固定化乙酰胆碱酯酶的反应器中，该酶将乙酰胆碱水解为胆碱。产物被第二种酶胆碱氧化酶转化为过氧化氢，通过安培法进行测定。使用了几种新型固定化技术，包括通过酶特异性抗体进行固定化，以固定这些酶以保持最大活性。当通过抗生物素蛋白-生物素连接固定酶时，观察到检测限有所改善。与将两种酶分别固定并用于两根柱中相比，当两种酶通过抗生物素蛋白-生物素连接一起固定在同一载体上时，获得了更好的灵敏度和检测限。柱后系统应用于脑组织提取物。
