Disposable electrochemical biosensor based on NAD(P)-dependent dehydrogenase and diaphorase

A method of making a disposable, single-use biosensor based on NAD(P)-dependent dehydrogenase and diaphorase, the method comprising:

• providing a sensing surface having at least a working electrode portion, a blank electrode portion, and a reference electrode portion;
• disposing a first soluble reagent on the working electrode portion, the first soluble reagent containing a NAD(P)-dependent dehydrogenase, NAD(P)+, diaphorase, and an oxidized form of a redox mediator and drying the first soluble reagent forming a first electrode matrix creating a working electrode, wherein the biosensor is configured such that the first soluble reagent dissolves while a sample fills a sample chamber of the biosensor, at least in part, by capillary action;
• disposing a second soluble reagent on the blank electrode portion, wherein the second soluble reagent and the first soluble reagent differ in composition at least in that the second soluble reagent does not contain dehydrogenase;
• and
• disposing a reference electrode material on the reference electrode portion forming a reference electrode, wherein the reference electrode material is one of Ag—AgCl or a reference electrode reagent containing at least a chemically oxidizing reagent selected from the group consisting of a reduced form of a redox mediator, an oxidized form of a redox mediator and a mixture of a reduced form and an oxidized form of a redox mediator and drying the reference electrode reagent forming a reference electrode matrix.