Analytical Sciences

Simple and Cost-effective Enzymatic Detection of Cholesterol Using Flow Injection Analysis

Murugan THIRUPPATHI,*1 Ching-Ying TSAI,*1 Tzu-Wen WANG,*2 Yu TSAO,*1 Tsung-Hung WU,*1 and Ja-an Annie HO*1,*2,*3,*4

*1 BioAnalytical Chemistry and Nanobiomedicine Laboratory, Department of Biochemical Science and Technology, National Taiwan University, Taipei 10617, Taiwan
*2 Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan
*3 Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan
*4 Center for Biotechnology, National Taiwan University, Taipei 10617, Taiwan

A flow-injection analytical (FIA) system was developed for the determination of cholesterol concentrations based on enzymatic reactions that occurred in a cholesterol oxidase (CHO_x)-immobilized, fused-silica capillary followed by electrochemical detection. The production of hydrogen peroxide from cholesterol in an enzymatic reaction catalyzed by CHO_x was subsequently oxidized electrochemically at an electrode. Our FlA system demonstrated its cost-effectiveness and utility at an applied potential of 0.6 V (vs. Ag/AgCl), a flow rate of 100 μL/min and, under optimal conditions, the resulting signal demonstrated a linear dynamic range from 50 μM to 1.0 mM with a limit of detection (LOD) of 12.4 μM, limit of quantification (LOQ) of 44.9 μM, and the coefficient of variation of 5.17%. In addition, validation of our proposed system using a reference HDL-cholesterol kit used for clinical diagnosis suggested our FIA system was comparable to commercial kits for the determination of the cholesterol incorporation amount in various aqueous liposomal suspensions. These good analytical features achieved by FIA could make the implementation of this methodology possible for on-line monitoring of cholesterol in various types of samples.

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