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

A direct, low-cost method to determine the concentration of lactose is an important goal with possible impact in various types of industry. In this study, a biosensor is reported that exploits the specific interaction between lactose and the enzyme beta-galactosidase (beta-Gal) normally employed to process lactose into glucose and galactose for lactose-intolerant people. The biosensor was made with beta-Gal immobilized in layer-by-layer (LbL) films with the polyelectrolyte poly(ethylene imine) (PEI) and poly(vinyl sufonate) (PVS) on an indium tin oxide (ITO) electrode modified with a layer of Prussian Blue (PB). With an ITO/PB/(PEI/PVS)(1)(PEI/beta-Gal)(30) architecture, lactose could be determined with an amperometric method with sensitivity of 0.31 mu A mmol(-1) cm(-2) and detection limit of 1.13 mmol L-1, which is sufficient for detecting lactose in milk and for clinical exams. Detection occurred via a cascade reaction involving glucose oxidase titrated as electrolytic solution in the electrochemical cell, while PB allowed for operation at 0.0 V versus saturated calomel electrode, thus avoiding effects from interfering species. Sum-frequency generation spectroscopy data for the interface between the LbL film and a buffer containing lactose indicated that beta-Gal lost order, which is the first demonstration of structural effects induced by the molecular recognition interaction with lactose.