Selective immobilization of biomolecules onto an activated polymeric adlayer

Abstract

The authors report a facile method for the selective immobilization of biomolecules onto a gold surface that was preactivated by a polymeric adlayer. The polymeric adlayer was designed to perform triple functions: high resistance to nonspecific protein adsorption, efficient surface anchoring, and subsequent covalent attachment of biomolecules. For this purpose, a random copolymer, poly(PEGMA-r-NAS), was synthesized by radical polymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and N-acryloxysuccinimide (NAS). In the first step, the polymeric adlayer was formed onto amine-terminated self-assembled monolayers (SAMs) on gold through covalent bond formation between reactive N-hydroxysuccinimide (NHS) ester of the copolymer and the amine of the SAMs. In the second step, amine-bearing biotin as a model biomolecule was covalently attached onto the polymeric adlayer that still contained unreacted NHS esters. The degrees of the binding sensitivity for a target protein and the nonspecific binding for four model proteins on the biotinylated polymeric adlayer were examined by surface plasmon resonance spectroscopy. Finally, the specific immobilization of rhodamin (TRITC)-conjugated streptavidin on the biotinylated polymeric adlayer was achieved by a simple microcontact printing technique, resulting in well-defined patterns of the protein.

Preparation of Protein A Membrane Adsorbers Using Strain-Promoted, Copper-Free Dibenzocyclooctyne (DBCO)-Azide Click Chemistry

“…Reaction of NHS ester with azido-PEG 3 -amine results in disappearance of the peaks assigned to the NHS carbonyl group and carbonate, and the appearance of peaks assigned to the amide group stretching at 1698 cm −1 , azide stretching at 2094 cm −1 , and CH 2 scissoring at 1558 cm −1 (spectrum C). Similar spectra have been reported for amine functionalized DSC surfaces [16].…”

Stamps, inks and substrates: polymers in microcontact printing

“…In another publication the same group used a polymer containing PEG as well as NHS functionality as a reactive coating on amino-SAMs on Au. 145 Part of the NHS groups served as covalent links to the amine surface. Residual NHS groups were then used to pattern amino-biotin by mCP which could afterwards be complexed by streptavidin.…”

Formation of activation‐free, selectively bioconjugatable poly(N‐acryloxysuccinimide‐co‐oligoethylene glycol methyl ether methacrylate) films by surface‐initiated ARGET ATRP

“…The selective conjugation with streptavidin on the biotinylated copolymeric films and the relevant S/N ratio were evaluated by measuring the adsorbed protein using ellipsometry and SPR. The optimal S/N ratio is 339.8, which is 69.3 times higher than the previously reported result ( S/N = 4.9) from an activation‐free method . The developed method in this study is also superior to the surface modification method with an activation step that resulted in 223.6 as S/N ratio .…”

“…For example, a reactive thin polymer film was fabricated as a platform for the immobilization of biomolecules on various substrates such as silicon wafer, glass, and gold substrates . We synthesized the copolymers with non‐biofouling and bioactive groups and grafted the copolymers onto silicon oxides and gold substrates by the formation of polymeric self‐assembled monolayers (SAMs) and amide coupling, respectively. While removing the activation step, it was found that the previously reported polymeric films had lower S/N ratios (defined as the ratio of specific binding capacity to non‐specific one) than DSC‐activated poly(OEGMA) films.…”

“…While removing the activation step, it was found that the previously reported polymeric films had lower S/N ratios (defined as the ratio of specific binding capacity to non‐specific one) than DSC‐activated poly(OEGMA) films. Using SPR spectroscopy, the S/N ratios of the activation‐free, copolymeric films and the DSC‐activated poly(OEGMA) films were found to be 4.9 and 223.6, respectively. The expansion of biosensor applications together with the increased interest in healthcare has enhanced the need to develop a facile, activation‐free surface modification method that can give high S/N ratio.…”