The invention provides methods for preparing a stable, multiple-use three
enzyme biosensor for the amperometric determination of
creatinine in biological liquids that has a useful lifetime that extends significantly beyond that of presently available amperometric biosensors. The
biosensor prepared by the methods of the invention encompasses a plurality of immobilized enzymes that are applied to the
biosensor as an
enzyme-
polymer composition. The enzymes, which can include
creatinine amidohydrolase,
creatine amidinohydrolase and
sarcosine oxidase, are immobilized into the
enzyme-
polymer composition simultaneously as well as applied to the biosensor simultaneously. Prior to being immobilized, the enzymes can be chemically modified by attaching one or more
polyethylene glycol (PEG) chains per enzyme
monomer. The
polymer component can be provided by a
polyurethane membrane. The invention also provides a method of preparing a biosensor that limits the
diffusion of silver ions emanating the
reference electrode, thereby preventing, contact between the silver ions and the enzymes. Related methods of preparing an enzyme-
polymer composition for incorporation into a
multiple use three enzyme biosensor for the amperometric determination of
creatinine in biological liquids also are provided. The invention also provides multiple-use biosensors and enzyme-polymer compositions prepared by the methods disclosed.