Glucose oxidase electrodes via reconstitution of the apo-enzyme:: tailoring of novel glucose biosensors

Reconstitution of an apo-flavoenzyme with a relay-FAD-cofactor dyad yields an electrically contacted enzyme, "electroenzyme". This is exemplified by the reconstitution of ape-glucose oxidase, apo-GOx, with a ferrocene-tethered FAD-cofactor. The resulting semisynthetic protein stimulates the bioelectrocatalyzed oxidation of glucose. Kinetic analysis of the ferrocene-FAD reconstituted enzyme reveals that its electrical communication with an electrode support is superior as compared to a protein randomly functionalized by ferrocene relay units. A pyrroloquinolinoquinone (PQQ)-FAD cofactor monolayer is assembled on a Au-electrode. Apo-GOx is reconstituted on the solid support to yield an aligned enzyme electrode of unprecendently efficient electrical contact. The electron-transfer turnover rate with the electrode is estimated to be 900+/-150 s(-1), a value close to the electron transfer rate between native GOx and molecular oxygen. The effective electrical communication of the integrated enzyme electrode stimulates the efficient bioelectrocatalyzed oxidation of glucose, and results in high current densities and high sensitivity for glucose. The effective electrical contact of the enzyme-electrode yields also a specific glucose sensing electrode that is not perturbed by oxygen or interferrants. The possible application of the enzyme-electrode as an invasive biosensor is addressed. (C) 1999 Elsevier Science B.V. All rights reserved.