An Integrated Amperometric Biosensor for the Determination of Lactose in Milk and Dairy Products

Abstract

An integrated amperometric biosensor for the determination of lactose is reported. The bioelectrode design is based on the use of a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode on which the enzymes beta-galactosidase (beta-Gal), glucose oxidase (GOD), peroxidase (HRP) and the mediator tetrathiafulvalene (TTF) are coimmobilized by a dialysis membrane. beta-Gal catalyzes the hydrolysis of lactose, and the produced glucose is catalytically oxidized to gluconic acid and H(2)O(2), which is reduced in the presence of HRP. This enzyme reaction is mediated by TTF, and the reduction of TTF(+) at 0.00 V (vs Ag/AgCl) gives rise to an amperometric signal proportional to the lactose concentration. The biosensor exhibits a good repeatability of the measurement carried out with the same biosensor, a good reproducibility of the responses obtained with different biosensors and a useful lifetime of 28 days. A linear calibration plot was obtained for lactose over the 1.5 x 10(-6) to 1.2 x 10(-4) M concentration range, with a limit of detection of 4.6 x 10(-7) M. The effect of potential interferents (sucrose, lactulose, fructose, arabinose, maltose, galactose, glucose and uric and ascorbic acids) on the biosensor response was evaluated. Furthermore, the bioelectrode exhibits a suitable performance in flow-injection systems in connection with amperometric detection. The developed biosensor was applied to the determination of lactose in milk and other foodstuffs (chocolate, butter, margarine, yogurt, cheese and mayonnaise), and the results obtained were validated by comparison with those provided by using a commercial enzyme test kit.

Determination of Lactose in Milk by Raman Spectroscopy

“…However, enzyme biosensors may be affected by the presence of galactose and glucose present in milk and milk products (Conzuelo et al 2010). In near-infrared spectroscopy methods, there are interferences from water absorption and spectral congestion limitations (Wallach and Oseroff 1974).…”

Recent advances in analytical techniques for the determination of lactose

“…Enzyme-based amperometric biosensors have been found to constitute versatile analytical devices with high selectivity that can be operated by unskilled personnel. Accordingly, they can be envisaged as serious competitors for conventional techniques, representing an attractive alternative for small industries [17][18][19].…”

Amperometric Detection of Lactose Using β-Galactosidase Immobilized in Layer-by-Layer Films

“…Examples include an amperometric biosensor with β-Gal, GOx, peroxidase (HRP), and the mediator tetrathiafulvalene (TTF) coimmobilized onto a gold electrode modified by a self-assembled monolayer (SAM). 2 Detection was carried out at 0.00 V (vs Ag/AgCl) with the signal being proportional to the lactose concentration between 1.5 and 120 μmol L −1 . Another amperometric biosensor was prepared by immobilizing β-Gal and GOx with glutaraldehyde onto a glassy carbon electrode coated with a mercury thin film.…”