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

• Published in: ACS applied materials & interfaces Vol. 6; no. 14; pp. 11657 - 11664
• Main Authors: Campos, Paula P, Moraes, Marli L, Volpati, Diogo, Miranda, Paulo B, Oliveira, Osvaldo N, Ferreira, Marystela
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 23.07.2014
• Subjects: Aspergillus oryzae - enzymology; beta-Galactosidase - chemistry; Biosensing Techniques - methods; Electrochemical Techniques - methods; Enzymes, Immobilized - chemistry; Fungal Proteins - chemistry; Glucose Oxidase - chemistry; Lactose - analysis; Membranes, Artificial; β-galactosidase; sum-frequency generation; amperometric detection; lactose; layer-by-layer
• ISSN: 1944-8244; 1944-8252
• DOI: 10.1021/am5024463

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.