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 α-chymotrypsin or urease is covalently linked to the PQQ-monolayer electrode. Biocatalyzed hydrolysis of N-acetyl-4-tyrosine ethyl ester (1) by α-chymotrypsin or biocatalyzed degradation of urea (2) by urease alters the pH of the electrolyte-solution. The changes in the pH are sensed by the redox-potential of the PQQ-redox-active units associated with the electrode. Tethering of electroactive pH-insensitive, ferrocene units to the protein enables the sensing of the pH variations by following the potential difference between PQQ and ferrocene electroactive units. This enables the use of the integrated PQQ-ferrocene-tethered enzyme electrode as a pH-controlled biosensor with an internal potentiai reference.