title: Amperometric detection of lactose using β-galactosidase immobilized in layer-by-layer films.
abstract: 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 β-galactosidase (β-Gal) normally employed to process lactose into glucose and galactose for lactose-intolerant people. The biosensor was made with β-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/β-Gal)30 architecture, lactose could be determined with an amperometric method with sensitivity of 0.31 μA 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 β-Gal lost order, which is the first demonstration of structural effects induced by the molecular recognition interaction with lactose.
authors.affiliation: Federal University of São Carlos, UFSCar, Campus Sorocaba , Rodavia João Leme dos Santos, km 110 - SP-264, São Carlos SP, 18052-780 Brazil.
authors.forename: Paula P
authors.initials: PP
authors.lastname: Campos
authors.forename: Marli L
authors.initials: ML
authors.lastname: Moraes
authors.forename: Diogo
authors.initials: D
authors.lastname: Volpati
authors.forename: Paulo B
authors.initials: PB
authors.lastname: Miranda
authors.forename: Osvaldo N
authors.initials: ON
authors.lastname: Oliveira
authors.forename: Marystela
authors.initials: M
authors.lastname: Ferreira
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology*; D015374:Biosensing Techniques / Q000379:methods*; D055664:Electrochemical Techniques / Q000379:methods; D004800:Enzymes, Immobilized / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry*; D005949:Glucose Oxidase / Q000737:chemistry; D007785:Lactose / Q000032:analysis*; D008567:Membranes, Artificial; D001616:beta-Galactosidase / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D008567:Membranes, Artificial; D005949:Glucose Oxidase; D001616:beta-Galactosidase; D007785:Lactose