Aldehyde-mediated protein-to-surface tethering via controlled diazonium electrode functionalization using protected hydroxylamines

We report a diazonium electro-grafting method for the covalent modification of conducting surfaces with aldehyde-reactive hydroxylamine functionalities that facilitate the wiring of redox-active (bio)molecules to electrode surfaces. Hydroxylamine monolayer formation is achieved via a phthalimide-protection and hydrazine-deprotection strategy that overcomes the multilayer formation that typically complicates diazonium surface modification. This surface modification strategy is characterized using electrochemistry (electrochemical impedance spectroscopy and cyclic voltammetry), X-ray photoelectron spectroscopy and quartz crystal microbalance with dissipation monitoring. Thus-modified glassy carbon, boron-doped diamond and gold surfaces are all shown to ligate to small molecule aldehydes, yielding surface coverages of 150-170, 40 and 100 pmol cm-2, respectively. Bio-conjugation is demonstrated via the coupling of a dilute (50 µM) solution of periodate-oxidized horseradish peroxidase enzyme to a functionalized gold surface under bio-compatible conditions (H2O solvent, pH 4.5, 25 °C).