Monolayers formed with N-hydroxysuccinimide (NHS) ester terminal groups are extensivelyused as coupling agents to link amine-containing biomolecules (e.g., proteins, peptides,and other molecular recognition elements) to surfaces for the fabrication of awide range of platforms in heterogeneous diagnostics. While this widely used methodfor immobilization simplifies biomolecule tethering to surfaces via amidelinkages, the hydrolysis of the terminal NHS group competes with amidization. Thispresentation details an investigation of this competition using succinimidylester-terminated monolayers on gold formed from disulfides (dithiobissuccinimidyl propionate, hexanoate, octanoate, and undecanoate) of variouschain lengths. Hydrolysis reactions were performed in borate buffer (pH 8.5)and other alkaline media; aminolysis reactions used ethylamine as a model atvarious concentrations and pH (8.5 to 10.5). The structure and reactivity ofthese adlayers were analyzed using infrared reflection spectroscopy (IRS), X-rayphotoelectron spectroscopy (XPS), electrochemical reductive desorption, andcontact angle measurements. Results show that the hydrolysis dominates theinterfacial reaction chemistry, with aminolysis only becoming competitive at highamine concentrations (>5.0 mM) and high pH. The implications of these findingsare discussed in terms of applying this form of coupling chemistry to proteinimmobilization.

Protein immobilization: Competing reactions in the formation of amide linkages via succinimidyl ester chemistry

246th National Meeting (2013)

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