Succinimidyl Ester Surface Chemistry: Implications of the Competition between Aminolysis and Hydrolysis on Covalent Protein Immobilization

Abstract

N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent protein immobilization in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer. This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh. More importantly, a projection of these heterogeneous kinetic rates to protein immobilization suggests that under coupling conditions in which low protein concentrations and buffers of near physiological pH are used, proteins are more likely physically adsorbed rather than covalently linked. This result is paramount for biosensors that use NHS chemistry for protein immobilization due to effects that may arise from noncovalently linked proteins.

Mixed‐monolayer of N‐hydroxysuccinimide‐terminated cross‐linker and short alkanethiol to improve the efficiency of biomolecule binding for biosensing

“…It was reported that similar to alkanethiols, disulfides are adsorbed on the gold surface spontaneously upon cleavage of the S‐S bond due to the reductive property of gold, resulting in the Au‐S bond formation. Complementary with these findings, S 2p spectra of DSP, NHSS and NHS derivative (disulfide containing molecules of similar structure) modified surfaces demonstrate the spontaneous cleavage of the S‐S bond and covalent attachment of these molecules through gold thiol (Au‐S) interactions without any treatment with reducing agents . The S 2p spectra of all the samples are shown in Figure .…”

“…All the O 1 s spectra have the characteristic oxygen peak around 532.0 eV which is partially attributed to environmental oxygen (Figure A). However, Lim et al, defined the peak at 532.1 eV as the C=O functional group of the DSP molecule . In addition, this group identified another peak at 534.4 eV, which corresponds to NHS ester oxygen.…”

“…The emergence of other peaks at different binding energies not present in blank gold surface, indicates the presence of functional groups of the molecules immobilized to surface. The peak at 288.6 eV and 286.2 eV in the C 1 s spectrum of DSP modified gold surface corresponds to C=O and C‐S respectively (Figure B). In a previous study, the 3,3′‐Dithiobissulfo‐succinimidylpropionate (DTSSP) (Sulfo‐DSP) immobilized gold surfaces were characterized by XPS .…”

Surface immobilization of functional molecules by reactive self‐assembling

“…These components were assigned to imide and thiadiazole groups and protonated imide group, respectively. [9,10] Also the XPS investigation of silane (Fig. 3d) revealed the presence of two components in the N 1s spectrum, localized at BE = 399.6 eV (1′′′) and 401.6 eV (2′′′) and assigned to amide or amine and protonated amide or amine groups, respectively.…”

Click Chemistry Immobilization of Antibodies on Polymer Coated Gold Nanoparticles

“…Most importantly, the coupling reaction is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry20 . Last but not least, the reaction between active esters and amino groups is notregiospecific and does not allow the controlled and oriented immobilization of the protein.…”