(187ag) Nhs Surfaces: Characterization and Protein Immobilization

Authors: 
Castner, D. G., University of Washington
Cheng, F., University of Washington
Dubey, M., University of Washington
Gamble, L. J., University of Washington
Grainger, D. W., University of Utah
Emoto, K., Accelr8 Technology Corporation


N-hydroxysuccinimide (NHS) esters are widely used as leaving groups to activate covalent coupling of amine-containing biomolecules onto surfaces in academic and commercial surface immobilizations. Their intrinsic hydrolytic instability is well-known and remains a concern for maintaining stable, reactive surface chemistry, especially for reliable longer-term storage. We have used x-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) to investigate surface hydrolysis in NHS-bearing organic thin films. Principal component analysis (PCA) of both positive and negative ion ToF-SIMS data was used to correlate changes in the well-defined NHS ester oligo(ethylene glycol) (NHS-OEG) self-assembled monolayers to their surface treatment. From PCA results, multi-variate peak intensity ratios were developed for monitoring NHS reactivity, thin film thickness and oxidation of the monolayers during surface hydrolysis. Aging in ambient air for up to seven days resulted in hydrolysis of some fraction of bound NHS groups, oxidation of some resident thiol groups, and deposition of adventitious hydrocarbon contaminants onto the monolayers. Overnight film immersion under water produced complete hydrolysis and removal of the NHS chemistry, as well as removal of some of the thiolated OEG chains. NHS regeneration of the hydrolyzed surfaces was assessed using the same multi-variable peak intensity ratio as well as surface coupling with amine-terminated molecules. Both aqueous and organic NHS regeneration methods produced surfaces with bound NHS concentrations approximately 50% of the bound NHS concentration on freshly prepared NHS-OEG monolayers. The metrics developed in this study provide methods for quantifying NHS chemistry on surfaces used for microarray, microfluidic, immunoassay, bioreactor, tissue engineering, and biomedical device fabrication. This work on model NHS-OEG monolayer system was then extended to commercial poly(ethylene glycol) (PEG)-based polymer films coated onto 1?x3? glass slides. NHS and methoxy-capped regions were co-patterned onto these slides using photolithographic methods, then imaged with ToF-SIMS/PCA. NHS surface reactive zones are clearly resolved at high sensitivity despite the complexity of the matrix chemistry. ToF-SIMS also detected the presence of photo-resist residue on the patterned surfaces. Surface-specific protein coupling was observed by surface-selective reaction of streptavidin with the NHS patterns. 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.