Oligosaccharide biosensor for direct monitoring of enzymatic activities using QCM-D

• Published in: Biosensors & bioelectronics Vol. 49; pp. 290 - 296
• Main Authors: Bouchet-Spinelli, Aurélie, Reuillard, Bertrand, Coche-Guérente, Liliane, Armand, Sylvie, Labbé, Pierre, Fort, Sébastien
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 15.11.2013; Elsevier
• Subjects: Bacillus - enzymology; Biological and medical sciences; Biosensing Techniques - methods; Biosensors; Biotechnology; Carbohydrate biosensor; Carbohydrate-active enzyme; Carbohydrates; Colorimetry; Enzyme Assays - methods; Enzymes; Fundamental and applied biological sciences. Psychology; Glycoside Hydrolases - analysis; Glycoside Hydrolases - metabolism; Glycosides; Humans; Methods. Procedures. Technologies; Microbalances; Monitoring; Oligosaccharides; Oligosaccharides - chemistry; Oligosaccharides - metabolism; Quartz Crystal Microbalance Techniques - methods; Quartz-crystal microbalance with dissipation monitoring (QCM-D); Self-assembled monolayers; Various methods and equipments; Quartz-crystal microbalance with dissipation monitoring (QCM-D); Carbohydrate biosensor; Carbohydrate-active enzyme; Oligosaccharides; Self-assembled monolayers; Self assembly; Monolayer; Enzyme; Oligosaccharide; Crystals; Enzymatic activity; Biosensor; Carbohydrate; Monitoring; Quartz microbalance
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2013.05.027

Enzymatic modification of saccharidic biomass is a subject of intensive research with potential applications in plant or human health, design of biomaterials and biofuel production. Bioengineering and metagenomics provide access to libraries of glycoside hydrolases but the biochemical characterization of these enzymes remains challenging, requiring fastidious colorimetric tests in discontinuous assays. Here, we describe a highly sensitive carbohydrate biosensor for the detection and characterization of glycoside hydrolases. Immobilization of oligosaccharides was achieved using copper-catalyzed azide-alkyne cycloaddition of maltoheptaose-modified probes onto self-assembled monolayers bearing azide reactive groups. This biosensor allowed detection of glycoside hydrolase activities at the picomolar level using quartz-crystal microbalance with dissipation monitoring (QCM-D). To our knowledge, this protocol provides the best performance to date for the detection of glycoside hydrolase activities. For each enzyme tested, we could determine the kinetic constant from the QCM-D data, and derive conclusions that correlated well with those of standard colorimetric tests. This opens the way to a new generation of rapid and direct tests characterizing functionally carbohydrate-active enzymes. •We describe a highly sensitive QCM-D biosensor for glycoside hydrolases detection.•The immobilization strategy combined efficiently the use of SAMs and CuAAC.•The detection of glycoside hydrolase activities reached the best detection level to date.•The test can distinguish endo and exo modes of action on a substrate.•The kinetic constants of the enzymes could be extrapolated from the QCM-D data.