Miniaturized Biosensors Employing Electropolymerized Permselective Films and Their Use for Creatinine Assays in Human Serum

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Abstract
Miniaturized, disposable amperometric biosensors for determination of creatinine in human serum are described. The base electrodes are fabricated using microelectronics techniques, to build a multilayer film structure on a polyimide foil. By using a thin electropolymerized film of poly(1,3-diaminobenzene), the electrochemical interferences from ascorbate, urate, acetaminophen, and other oxidizable species are greatly diminished. The multienzyme system (creatininase, creatinase, sarcosine oxidase) is immobilized on top of the permselective layer using cross-linking of the proteins with glutaraldehyde. The electropolymerization conditions for obtaining almost ideal permselectivity of the inner layer are defined, as well as the optimal enzyme layer preparation. A composite polymeric outer membrane [Nafion + poly-(2-hydroxyethyl methacrylate)] is used for diffusion control and to protect the enzyme layer from fouling. The reagentless planar sensors for creatinine and creatine have fast response time (t95 = 1 min), linear response up to 1.2 mM in batch-type and 2.0 mM in flow injection analysis and a detection limit of 10−20 μM. They are applied in a differential setup for creatinine assay in control and hospital human serum samples and are suitable for incorporation in a portable analyzer.