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

Reconstitution of an apo-flavoenzyme with a relay-FAD-cofactor dyad yields an electrically contacted enzyme, "electroenzyme". This is exemplified by t he reconstitution of ape-glucose oxidase, apo-GOx, with a ferrocene-tethere d FAD-cofactor. The resulting semisynthetic protein stimulates the bioelect rocatalyzed oxidation of glucose. Kinetic analysis of the ferrocene-FAD rec onstituted enzyme reveals that its electrical communication with an electro de support is superior as compared to a protein randomly functionalized by ferrocene relay units. A pyrroloquinolinoquinone (PQQ)-FAD cofactor monolay er is assembled on a Au-electrode. Apo-GOx is reconstituted on the solid su pport to yield an aligned enzyme electrode of unprecendently efficient elec trical contact. The electron-transfer turnover rate with the electrode is e stimated to be 900+/-150 s(-1), a value close to the electron transfer rate between native GOx and molecular oxygen. The effective electrical communic ation of the integrated enzyme electrode stimulates the efficient bioelectr ocatalyzed oxidation of glucose, and results in high current densities and high sensitivity for glucose. The effective electrical contact of the enzym e-electrode yields also a specific glucose sensing electrode that is not pe rturbed 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.