Eur Food Res Technol (1999) 209 : 220–226 Q Springer-Verlag 1999 ORIGINAL PAPER N. Adányi 7 E.E. Szabó 7 M. Váradi Multi-enzyme biosensors with amperometric detection for determination of lactose in milk and dairy products Received: 24 August 1998 / Revised version: 9 November 1998 N. Adányi (Y) 7 E.E. Szabó 7 M. Váradi Central Food Research Institute, Herman O. út 15, H-1022, Budapest, Hungary e-mail: h9807ada6ella.hu Abstract In home-made sensors coimmobilizing en- zymes in thin-layer plexi-cells on natural protein mem- branes, three enzyme cells: b-galactosidase and galac- tose oxidase (A), b-galactosidase and glucose oxidase (B) and b-galactosidase, galactose oxidase and glucose oxidase (C) were built into a flow-injection-analyzer system. The lactose was decomposed and oxidized by the immobilized enzymes and the hydrogen peroxide generated during the enzymatic reactions was deter- mined by amperometric detection. The parameters for biochemical and electrochemical reactions (concentra- tion of buffer, temperature, flow rate) were optimized in each enzyme cell. The pH optima of the lactose measurement was determined in the three enzyme cells mentioned above. The pH optimum of the cells A, B and C were 6.4, 4.5 and 4.8, respectively. The measur- ing ranges were 1–5 mM, 2–10 mM and 1–5 mM, while the detection limits were 0.5, 1.0 and 0.5 mM, respec- tively. More than 600, 1000 and 800 samples could be measured with these cells, respectively. Commercial milk and instant dessert powder products were ana- lysed also. Our results showed that the cells B and C were more suitable for the determination of the lactose content of milk. For samples of dairy products contain- ing added glucose, starch and other carbohydrates, en- zyme cell A could be used for the efficient determina- tion of lactose in one step. Key words Lactose determination 7 Amperometric biosensor 7 Thin-layer enzyme cell Introduction The determination of lactose in milk and milk products is routinely carried out in the dairy industry to ensure effective process and product control. Several methods are available for this purpose, including gas and liquid chromatography, polarimetry, gravimetry and spectro- photometry using alkaline methylamine. Recently, the application of immobilized enzymes has given a powerful new process in the analytical field. Coupling of immobilized enzymes with electrochemical sensors provides devices that can be used for rapid, re- petitive and cheap assays. For the construction of biosensors, enzymes can be immobilized on the surface of the measuring electrode or on the surface of different resins or controlled-pore glass used in analytical reactors. Volesky and Emond [1] constructed a specific continuous-flow analytical system for the potentiometric determination of lactose. b-galactosidase and glucose oxidase immobilized on a phenol-formaldehyde resin were employed in analytical column. The time delay of the measurement was ap- proximately 15 min. Watanabe et al. [2] made sensors for the simultaneous determination of different carbo- hydrates using a combination of enzymes and a Clark- type oxygen electrode. Three sheets of triacetate cellu- lose membrane covered by the enzymes were placed on the Teflon membrane of the oxygen electrode and cov- ered with a dialysis membrane. Several studies have re- ported on amperometric detection after enzymatic reactions. Lundbäck and Olsson [3] developed a sensor for galactose, lactose and dihidroxyacetone (DHA) us- ing galactose oxidase in a flow-injection-analyzer (FIA) system with an immobilized enzyme reactor. The en- zyme was immobilized on aryloamino-derivatized con- trolled-pore glass, and packed into a reactor. Mascini [4] described an amperometric method in which the en- zymes were immobilized on the surface of a platinum electrode, two dialysis membranes fixed with an O-ring serving as the support. Pilloton and Mascini [5] pre-