An enzyme electrode for extended linearity citrate measurements based on modified polymeric membranes

Pyruvate oxidase (POD), oxaloacetate decarboxylase (AOCD), and citrate lyas e (CL) were coimmobilized (or immobilized separately) in a series of polyme ric membranes for the construction of amperometric bisensors of pyruvic, ox aloacetic, or citric acid in concentrated samples. For oxaloacetic acid, PO D, and OACD were coimmobilized on dialysis membranes and were used in a mul timembrane configuration with an inner spin-coated cellulose acetate (CA) m embrane modified with isopropyl myristate (IPM) and an outer diffusion rest ricted membrane of cellulose acetate modified with a cationic surfactant. T hese membranes were also tested in a POD laminate containing the cofactors FAD and TPP for monitoring the response stability with duration of exposure to an external electrolyte and found to be effective in reagentless mode, with good pyruvate response stability over 3 h without compromise of signal size. Coimmobilization of POD/OACD and CL on different types of high prote in binding membrane such as mixed cellulose ester (HA) was investigated. Th e relative optimum concentrations of several activators (divalent cations) and cofactors such as FAD and thiamine pyrophosphate (TPP) were investigate d with the probe assembled as a pyruvate biosensor. An extended linearity u p to 100 mM citric acid was achieved.