Flow-through multi-enzyme electrodes for the determination of lactose

Abstract

Four types of lactose-sensing electrodes based on uni-, di-, tri- and tetra-enzyme systems have been studied. The appropriate combinations of enzymes [lactase (L), glucose oxidase (G), mutarotase (M) and galactose oxidase (Ga)] were chemically immobilised on nylon net which was placed over a platinum electrode housed in a three-electrode Stelte micro-cell modified for flow injection. Lactose was determined amperometrically by monitoring the hydrogen peroxide enzymolysis product at +600 mV versus a silver-silver chloride reference electrode. The strengths of signals from six different lactose electrodes based on combinations of the four enzymes decreased in the order: LMG > LMGGa > LG > LGGa > LGa > Ga. The expected two-fold increase in sensitivity from the tri-enzyme electrode, LGGa, and the tetra-enzyme electrode, LMGGa, over the tri-enzyme electrode, LMG, did not materialise. Rather, the LMG electrode was superior in terms of lactose response and linear range (3 × 10^–6–2 × 10^–3M). In addition, the LMG electrode also exhibited short response times (15–20 s), high resistance to temperature, a long lifetime (only a 5% reduction in signals after 18 h continuous flow of 1 mM lactose) and good storage stability (ca. 8 months in 0.1 M, pH 8 phosphate buffer at 4 °C) with intermittent use. Data for the determination of lactose in foodstuffs with the enzyme electrode were comparable to those obtained using a soluble enzyme test kit (Boehringer Mannheim UV method).