An Enzyme Electrode for Extended Linearity Citrate Measurements Based on Modified Polymeric Membranes

Abstract

Pyruvate oxidase (POD), oxaloacetate decarboxylase (AOCD), and citrate lyase (CL) were coimmobilized (or immobilized separately) in a series of polymeric membranes for the construction of amperometric bisensors of pyruvic, oxaloacetic, or citric acid in concentrated samples. For oxaloacetic acid, POD, and OACD were coimmobilized on dialysis membranes and were used in a multimembrane configuration with an inner spin-coated cellulose acetate (CA) membrane modified with isopropyl myristate (IPM) and an outer diffusion restricted membrane of cellulose acetate modified with a cationic surfactant. These 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 protein binding membrane such as mixed cellulose ester (HA) was investigated. The relative optimum concentrations of several activators (divalent cations) and cofactors such as FAD and thiamine pyrophosphate (TPP) were investigated with the probe assembled as a pyruvate biosensor. An extended linearity up to 100 mM citric acid was achieved.