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Abstract
A bienzymatic sensing layer containing two enzymes able to work sequentially, choline oxidase (ChOD) and phospholipase D (PLaseD), was used to design an electrochemical biosensor for the detection of either a water-soluble (choline) or insoluble (phosphatidylcholine) substrate. A photocrosslinkable polymer, poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ), was used as host-matrix for enzyme immobilization. Controlled amounts of PVA-SbQ and of the two enzymes were directly coated on a platinum disk, then photopolymerized. The compatibility of working conditions for choline and phosphatidylcholine detection in the presence of Triton X-100 and CaCl2 was investigated. The effect of the activity ratio PLaseD / ChOD on the sensor performance was determined. The sensitivities to choline and to phosphatidylcholine were 18 mA.1mol−1 and 0.66 mA.1.mol−1 respectively, the detection limit being 1.5.10−8 M for choline and 1.5.10−6 M for phosphatidylcholine. The linear range extended up to ca. 10−4 M for choline and ca. 2.10−5 M for phosphatidylcholine and the response time was close to 30 seconds for choline and ca. 2 min for phosphatidylcholine.