Immobilization of the Enzyme β-Lactamase on Biotin-Derivatized Poly(l-lysine)-g-poly(ethylene glycol)-Coated Sensor Chips:  A Study on Oriented Attachment and Surface Activity by Enzyme Kinetics and in Situ Optical Sensing

Abstract

Understanding the conformation, orientation, and specific activity of proteins bound to surfaces is crucial for the development and optimization of highly specific and sensitive biosensors. In this study, the very efficient enzyme beta-lactamase is used as a model protein. The wild-type form was genetically engineered by site-directed mutagenesis to introduce single cysteine residues on the surface of the enzyme. The cysteine thiol group is subsequently biotinylated with a dithiothreitol (DTT)-cleavable biotinylation reagent. beta-Lactamase is then immobilized site-specifically via the biotin group on neutral avidin-covered surfaces with the aim to control the orientation of the enzyme molecule at the surface and study its effect on enzymatic activity using Nitrocefin as the substrate. The DTT-cleavable spacer allows the release of the specifically bound enzyme from the surface. Immobilization of the enzyme is performed on a monolayer of the polycationic, biotinylated polymer PLL-g-PEG/PEG-biotin assembled on niobium oxide (Nb2O5) surfaces via neutral avidin as the docking site. Two different assembly protocols, the sequential adsorption of avidin and biotinylated beta-lactamase and the immobilization of preformed complexes of beta-lactamase and avidin, are compared in terms of immobilization efficiency. In situ optical waveguide lightmode spectroscopy and colorimetric analysis of enzymatic activity were used to distinguish between specific and unspecific enzyme adsorption, to sense quantitatively the amount of immobilized enzyme, and to determine Michaelis-Menten kinetics. All tested enzyme variants turned out to be active upon immobilization at the polymeric surface. However, the efficiency of immobilized enzymes relative to the soluble enzymes was reduced about sevenfold, mainly because of impaired substrate (Nitrocefin) diffusion or restricted accessibility of the active site. No significant effect of different enzyme orientations could be detected, probably because the enzymes were attached to the surface through long, flexible PEG chain linkers.

Towards area‐based in vitro metabolic engineering: Assembly of Pfs enzyme onto patterned microfabricated chips

“…To enhance biocompatibility, researchers have created polymer interfaces between the enzymes and the device surfaces. [2][3][4][5][6][7][8][9] The next generation of ''smart'' biomaterials will require the intimate coupling of advances in microfabrication and biomolecular recognition. The novelty of the approach described here is the use of patterned microdevices (conductive surface patterning) for the assembly of enzymes through an activatable ''pro-tag.''…”

Formation and removal of alkylthiolate self-assembled monolayers on gold in aqueous solutions

“…[7][8][9][10] In practice, SAMs employing biotin have been studied in association with streptavidin conjugated to DNA, proteins, and nanoparticles. [11][12][13][14][15][16] Such SAMs are vital for bioassay technologies such as DNA chips, protein chips, and small molecule biosensors.…”

Electrochemical Biosensors - Sensor Principles and Architectures

“…Donors for the reaction with HRP are molecules such as phenols, aromatic amines, thioaminosoles or iodide [208]. Other, less commonly used enzymes comprise beta-lactamase [209], urea and urease [10].…”