A highly sensitive sensor for the amperometric assay of glucose in aqueous media having a face-to-face sandwich configuration is provided which comprises a sensing electrode strip containing a first redox mediator in an electrically conductive coating and a reference electrode strip in simultaneous contact with a reagent strip containing a second redox mediator which is sandwiched between the active electrode surfaces. An opening is provided in the reference electrode for the introduction of samples. In one embodiment, a whole blood separation membrane is interposed between the reference electrode and the reagent strip to filter red blood cells and other particles from whole blood to enable direct assay of glucose without sample preparation. The sensing electrode comprises a non-conductive support member having an electrically conductive layer containing the first redox mediator. The reference electrode is typically a Ag/AgCl electrode formed by coating an ink containing Ag/AgCl dispersed in a resin on a non-conductive base. The reagent strip is a porous or fibrous carrier, typically a paper, impregnated with a mixture containing the second redox mediator, glucose oxidase, horseradish peroxidase, at least one surfactant and at least one stabilizer comprising an aqueous thickening agent. In one version of the sensor, the sensing electrode comprises a support member of polyester film coated with an electrically conductive graphite composition containing dimethylferrocene as the first redox mediator and the second redox mediator comprises potassium ferrocyanide.
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