Regio- and Chemoselective Immobilization of Proteins on Gold Surfaces

Abstract

Protein chips are powerful tools as analytical and diagnostic devices for detection of biomolecular interactions, where the proteins are covalently or noncovalently attached to biosensing surfaces to capture and detect target molecules or biomarkers. Thus, fabrication of biosensing surfaces for regio- and chemoselective immobilization of biomolecules is a crucial step for better biosensor performance. In our previous studies, a regio- and chemoselective immobilization strategy was demonstrated on glass surfaces. This strategy is now used to regioselectively attach proteins to self-assembled monolayers (SAMs) on gold surfaces. Recombinant green fluorescent protein (GFP), glutathione S-transferase (GST), and antibody-binding protein G, bearing a C-terminal CVIA motif, were prepared and a farnesyl analogue with an ω-alkyne moiety was attached to the sulfhydryl moiety in the cysteine side chain by protein farnesyltransferase. The proteins, modified with the bioorthogonal alkyne functional group, were covalently and regioselectively immobilized on thiol or dithiocarbamate (DTC) SAMs on a gold surface by a Huigsen [3 + 2] cycloaddition reaction with minimal nonspecific binding. A concentration-dependent increase of fluorescence intensity was observed in wells treated with GFP on both thiol- and DTC-SAMs. The highly ordered, densely packed layer allowed for a high loading of immobilized protein, with a concomitant increase in substrate binding capacity. The DTC-SAMs were substantially more resistant to displacement of the immobilized proteins from the gold surface by β-mercaptoethanol than alkane-thiol SAMs.

Microelectrical Impedance Spectroscopy for the Differentiation between Normal and Cancerous Human Urothelial Cell Lines: Real-Time Electrical Impedance Measurement at an Optimal Frequency

“…There are other cell-capturing methods, such as chemical and electrical immobilization techniques. Chemical immobilization techniques have been utilized to trap ligands, proteins, and cells [ 19 – 21 ]. Although this method shows stable binding of the targets on a surface, the preliminary process of surface treatment using adhesion materials is necessary to immobilize the targets.…”

Protein Prenylation: Enzymes, Therapeutics, and Biotechnology Applications

“… 119 Recently, Poulter and co-workers achieved highly ordered, regioselective immobilization of the glutathione S-transferase enzyme and antibody-binding protein G to self-assembled monolayers on gold surfaces. 120 They further created sandwich antibody arrays of immobilized recombinant antibody-binding protein L for capturing antibodies for direct- and sandwich-type immunofluorescent detection of ligands in a microarray format. 121 In general, the prenylation-based immobilization strategy has several potential biomedical and biotechnology applications where oriented protein immobilization is required, such as protein arrays, and diagnostic applications based on immunoassays, surface plasmon resonance (SPR), or electrochemical methods.…”

Perspective: A toolbox for protein structure determination in physiological environment through oriented, 2D ordered, site specific immobilization

“…Six of those classes are particularly suited for the purpose of anchoring: catalyzed cyclo-addition, 20,21 modified Staudinger ligation, 22,23 Diels-Alder cyclo-addition, 24,25 thiol-ene additions, 26,27 oxime ligation, 28,29 and Expressed Protein ligation, such as Split-intein-mediated ligation 30,31 or farnesyltransferase-related methods 32 . While these are not strictly single chemical reactions, they are used to bind a POI to a surface by means of the naturally occurring protein trans-splicing process 33 (split-intein mediated case) or farnesyltransferase mediated binding 19 …”