Development of organic phase amperometric biosensor for measuring cholesterol in food samples

Abstract

Previous works on organic phase enzyme electrodes (OPEE) applied methods that worked mostly under stirred conditions. The aim of our present work was to develop a flow-through measuring set-up for determination of cholesterol content in organic media. For determination of free cholesterol content cholesterol oxidase (COD) was used, while for measuring the total cholesterol content a bi-enzyme cell containing immobilised cholesterol esterase (CE) and cholesterol oxidase was developed. Enzyme immobilisation took place on a natural protein membrane, by a glutaraldehyde cross-linking method, in a thin-layer enzyme cell made from Teflon. The enzyme cell was connected into a stopped-flow injection system (SFIA), with a flow-through amperometric detector. The parameters of biochemical and electrochemical reactions were measured. The effect on amperometric detection of different organic salts as electron mediator or conducting salts was studied. The optimal concentration of (TBATS) was 2.4 mg L⁻¹ while for (FMCA) an optimal concentration was found at 0.4 mg L⁻¹. The minimum amount of water, necessary for enzymatic activity in the organic phase, was also determined. Changing the concentration of toluene in acetonitrile carrier solution, the peaks increased definitely in the range 10–40% toluene. Since CE and COD were immobilised together in the enzyme cell, the conversion rate was found to be about 0.7–0.8 when the toluene content was higher then 30%. The linear measuring range for cholesterol oleate and cholesterol was 0.1–0.5 mM. Total cholesterol content of lard, butter and pasta samples were determined. It is concluded that an organic phase bi-enzyme cell may be suitable for cholesterol determination in food.