Dataset Information

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.

SUBMITTER: Lim CY

PROVIDER: S-EPMC4222659 | biostudies-other | 2014 Nov

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Many chemical routes have been proposed to immobilize peptides on biomedical device surfaces, and in particular, on dental implants to prevent peri-implantitis. While a number of factors affect peptide immobilization quality, an easily controllable factor is the chemistry used to immobilize peptides. These factors affect peptide chemoselectivity, orientation, etc., and ultimately control biological activity. Using many different physical and chemical routes for peptide coatings, previous research has intensely focused on immobilizing antimicrobial elements on dental implants to reduce infection rates. Alternatively, our strategy here is different and focused on promoting formation of a long-lasting biological seal between the soft tissue and the implant surface through transmembrane, cell adhesion structures called hemidesmosomes. For that purpose, we used a laminin-derived call adhesion peptide. However, the effect of different immobilization chemistries on cell adhesion peptide activity is vastly unexplored but likely critical. Here, we compared the physiochemical properties and biological responses of a hemidesmosome promoting peptide immobilized using silanization and copper-free click chemistry as a model system for cell adhesion peptides. Successful immobilization was confirmed with water contact angle and X-ray photoelectron spectroscopy. Peptide coatings were retained through 73 days of incubation in artificial saliva. Interestingly, the non-chemoselective immobilization route, silanization, resulted in significantly higher proliferation and hemidesmosome formation in oral keratinocytes compared to chemoselective click chemistry. Our results highlight that the most effective immobilization chemistry for optimal peptide activity is dependent on the specific system (substrate/peptide/cell/biological activity) under study. Overall, a better understanding of the effects immobilization chemistries have on cell adhesion peptide activity may lead to more efficacious coatings for biomedical devices.

Project description:High-fidelity surface functional group (e.g., N-hydroxysuccinimide (NHS) reactive ester) patterning is readily and reliably achieved on commercial poly(ethylene glycol) (PEG)-based polymer films already known to exhibit high performance non-fouling properties in full serum and in cell culture conditions. NHS coupling chemistry co-patterned with methoxy-capped PEG using photolithographic methods is directly spatially imaged using imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) and principal components statistical analysis. Patterned NHS surface reactive zones are clearly resolved at high sensitivity despite the complexity of the polymer matrix chemistry. ToF-SIMS imaging also reveals the presence of photo-resist residue remaining from typical photolithography processing methods. High cross-correlation between various ion-derived ToF-SIMS images is observed, providing sensitive chemical corroboration of pattern chemistry and biological reactivity in complex milieu. Surface-specific protein coupling is observed first by site-selective reaction of streptavidin with NHS patterns, followed by identical patterns of biotinylated Alexa-labeled albumin coupling. This suggests that streptavidin immobilized on the patterns remains bioactive. Fluorescently labeled full serum is shown to react selectively with NHS-reactive regions, with minimal signal from methoxy-capped regions. Insufficient serum is adsorbed under any conditions to these surfaces to support cell attachment in serum-containing media. This reflects the high intrinsic non-adsorptive nature of this chemistry. Fibroblasts attach and proliferate in serum culture only when a cell adhesion peptide (RGD) is first grafted to NHS regions on the PEG-based surfaces. Longer-term serum-based cell culture retains high cell-pattern fidelity that correlates with chemical imaging of both the NHS and RGD patterns and also lack of cell adhesion to methoxy-capped regions. Cell staining shows orientation of adherent cells within the narrow patterned areas. Cell patterns are consistently retained beyond 15 days in serum media.

Dataset Information

Succinimidyl ester surface chemistry: implications of the competition between aminolysis and hydrolysis on covalent protein immobilization.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets