Biosensors with amperometric detection of enzymatically controlled pH-changes

New biosensors based on amperometric detection of enzymatically controlled pH-changes are described. Pyrroloquinoline quinone (PQQ) is assembled as a monolayer onto a Au-electrode, and alpha-chymotrypsin or urease is covalent ly linked to the PQQ-monolayer electrode. Biocatalyzed hydrolysis of N-acet yl-4-tyrosine ethyl ester (1) by alpha-chymotrypsin or biocatalyzed degrada tion of urea (2) by urease alters the pH of the electrolyte solution. The c hanges in the pH are sensed by the redox-potential of the PQQ-redox-active units associated with the electrode. Tethering of electroactive pH-insensit ive, Ferrocene units to the protein enables the sensing of the pH variation s by following the potential difference between pQQ and ferrocene electroac tive units. This enables the use of the integrated PQQ-ferrocene-tethered e nzyme electrode as a pH-controlled biosensor with an internal potential ref erence.