Cholesterol oxidase modified microelectrodes for detection of cholesterol in the plasma membrane of single cells.

摘要

Cholesterol oxidase was immobilized on electrode supported lipid bilayer membranes in an active state. Lipid molecules with a thiol functionality were used to form a sub-monolayer covalently linked to either indium tin oxide (ITO) or platinum electrode surfaces. The outer leaflet of the lipid bilayer membrane was formed using a deoxycholate dialysis method for modification of ITO electrodes. The vesicle fusion method was used to deposit the outer lipid leaflet on platinum electrode surfaces. Ferrocyanide/ferricyanide cyclic voltammetry was used to qualitatively monitor the formation of lipid bilayer at the electrode surfaces. Amperometry was used to detect hydrogen peroxide produced during the enzymatic oxidation of cholesterol at room temperature. Flow injection analysis of solution phase cholesterol at oxidase modified ITO, conventionally sized platinum electrodes, and platinum microelectrodes are reported. Cholesterol oxidase modified platinum electrodes showed Michaelis-Menten kinetic behavior for oxidation of solution phase cholesterol. The characterization studies using solution phase cholesterol at microelectrodes also suggested that the electrode response is limited by enzyme kinetics. Steady state current responses are obtained when the enzyme modified platinum microelectrodes are positioned in contact with the lipid membrane of giant vesicles containing cholesterol as a membrane constituent. The electrode responses correlated with the cholesterol content of the vesicle membrane where higher cholesterol content produces larger responses. Data obtained on contacting Xenopus oocytes demonstrated detection of cholesterol present in the cell plasma membrane. When the membrane cholesterol was decreased by exposure to cyclodextrin solution, decreased amperometric response were observed. For studies at 37 °C, cholesterol oxidase was covalently linked to platinum microelectrodes modified with a sub-monolayer of 11-mercapto undecanoic acid (MUA) using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) as a cross-linker. Covalently modified oxidase electrodes were used to detect cholesterol in the plasma membrane of oocytes and in the macrophage plasma membrane at 37 °C. Also reported are tapping mode - scanning force microscopy of cholesterol oxidase immobilized in a lipid membrane on mica. Topographic and phase contrast images are consistent with monomers and aggregates of cholesterol oxidase molecules immobilized in the lipid bilayer membrane.