Glucose minisensor based on self-assembled biotinylated phospholipid membrane on a solid support and its physical properties

Abstract

We have developed a glucose minisensor based on a biotinylated, supported phospholipid membrane (s-BLM). We immobilized glucose oxidase (GOX) by coupling an avidin-GOX complex to a phospholipid bilayer formed from biotinylated crude ox brain extract (COB). The bilayer was supported on the free metal tip of a Teflon-coated stainless steel wire (diameter, 0.33 mm). The determination of glucose was based on detection of enzymatically generated hydrogen peroxide at a potential of +670 mV. We found an almost linear increase of membrane current with increasing glucose concentration up to 10 mM and a saturation effect above 30 mM glucose. A lower voltage for detection of glucose was made possible by modification of the membrane by the electron carrier TCNQ. Several methods have been used to study the physical properties of native and modified s-BLM and conventional BLM. With the electrostriction method we showed that addition of avidin-GOX complex to the electrolyte in which the biotinylated s-BLM was formed resulted in a decrease of membrane capacitance and a decrease of membrane compressibility perpendicular to the bilayer surface. The capacitance relaxation method was used to determine the changes of dielectric relaxation times (reorientation of dipole moments of polar groups of individual lipid molecules or lipid clusters) following addition of avidin-GOX. Native BLM formed from COB extract (2% solution in n-decane: butanol (8:1 v/v) exhibited one relaxation time of (5 ± 1) μs. Additional relaxation components (115 ± 27 μs and 26 ± 1 μs) appeared in BLM modified by biotin. Addition of the avidin-GOX complex to the biotinylated BLM resulted in the appearance of a slow component 505 ± 16 m ̊ s . These results clearly document the interaction of the strongly immobilized enzyme with the bilayer.