Multienzymatic-rotating biosensor for total cholesterol determination in a FIA system

Fabrication of an amperometric-rotating biosensor for the enzymic detn. of cholesterol is reported. The assay utilizes a combination of three enzymes: cholesterol esterase (ChE), cholesterol oxidase (ChOx) and peroxidase (HRP); which were co-immobilizing on a rotatory disk. The method is developed by the use of a glassy carbon electrode as detector vs. Ag/AgCl/3 M NaCl in conjunction with a sol.-redox mediator 4-tert-butylcatechol (TBC). ChE converts esterified cholesterol to free cholesterol, which is then oxidized by ChOx with hydrogen peroxide as product. TBC is converted to 4-tert-butylbenzoquinone (TBB) by hydrogen peroxide, catalyzed by HRP, and the glassy carbon electrode responds to the TBB concn. The system has integrated a micro packed-column with immobilized ascorbate oxidase (AAOx) that works as prereactor to eliminate L-ascorbic acid (AA) interference. This method could be used to det. total cholesterol concn. in the range 1.2 micro M-1 mM (r = 0.999). A fast response time of 2 min has been obsd. with this amperometric-rotating biosensor. Lifetime is up to 25 days of use. The calcd. detection limits was 11.9 nM. Reproducibility assays were made using repetitive stds. solns. (n = 5) and the percentage std. error was less than 4 . [on SciFinder (R)]