Immobilization of peroxidase glycoprotein on gold electrodes modified with mixed epoxy-boronic Acid monolayers.
The development of bioelectronic enzyme applications requires the immobilization of active proteins onto solid or colloidal substrates such as gold. Coverage of the gold surface with alkanethiol self-assembled monolayers (SAMs) reduces nonspecific adsorption of proteins and also allows the incorporation onto the surface of ligands with affinity for complementary binding sites on native proteins. We present in this work a strategy for the covalent immobilization of glycosylated proteins previously adsorbed through weak, reversible interactions, on tailored SAMs. Boronic acids, which form cyclic esters with saccharides, are incorporated into SAMs to weakly adsorb the glycoprotein onto the electrode surface through their carbohydrate moiety. To prevent protein release from the electrode surface, we combine the affinity motif of boronates with the reactivity of epoxy groups to covalently link the protein to heterofunctional boronate-epoxy SAMs. The principle underlying our strategy is the increased immobilization rate achieved by the weak interaction-induced proximity effect between slow reacting oxyrane groups in the SAM and nucleophilic residues from adsorbed proteins, which allows the formation of very stable covalent bonds. This approach is exemplified by the use of phenylboronates-oxyrane mixed monolayers as a reactive support and redox-enzyme horseradish peroxidase as glycoprotein for the preparation of peroxidase electrodes. Quartz crystal microbalance, atomic force microscopy, and electrochemical measurements are used to characterize these enzymatic electrodes. These epoxy-boronate functional monolayers are versatile, stable interfaces, ready to incorporate glycoproteins by incubation under mild conditions.
Abad
José M
JM
Instituto de Catálisis y Petroleoquímica and Instituto de Ciencia de Materiales de Madrid, CSIC, Campus Universidad Autónoma de Madrid, Spain.
Vélez
Marisela
M
Santamaría
Carolina
C
Guisán
Jose M
JM
Matheus
Pedro R
PR
Vázquez
Luis
L
Gazaryan
Irina
I
Gorton
Lo
L
Gibson
Tim
T
Fernández
Víctor M
VM
D000327:Adsorption; D001897:Boronic Acids / Q000737:chemistry*; D004563:Electrochemistry; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry*; D004852:Epoxy Compounds / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry*; D007700:Kinetics; D018625:Microscopy, Atomic Force; D013816:Thermodynamics
D001897:Boronic Acids; D004800:Enzymes, Immobilized; D004852:Epoxy Compounds; D006023:Glycoproteins; C028592:3-aminobenzeneboronic acid; D006046:Gold; D006735:Horseradish Peroxidase