| Abstract
| - N-Hydroxysuccinimide (NHS) esters are widely used asleaving groups to activate covalent coupling of amine-containing biomolecules onto surfaces in academic andcommercial surface immobilizations. Their intrinsic hydrolytic instability is well-known and remains a concernfor maintaining stable, reactive surface chemistry, especially for reliable longer term storage. In this work, weuse X-ray photoelectron spectroscopy and time-of-flightsecondary ion mass spectrometry (TOF-SIMS) to investigate surface hydrolysis in NHS-bearing organic thinfilms. Principal component analysis (PCA) of both positiveand negative ion TOF-SIMS data was used to correlatechanges in the well-defined NHS ester oligo(ethyleneglycol) (NHS-OEG) self-assembled monolayers to theirsurface treatment. From PCA results, multivariate peakintensity ratios were developed for monitoring NHSreactivity, thin-film thickness, and oxidation of the monolayers during surface hydrolysis. Aging in ambient air forup to 7 days resulted in hydrolysis of some fraction ofbound NHS groups, oxidation of some resident thiolgroups, and deposition of adventitious hydrocarbon contaminants onto the monolayers. Overnight film immersionunder water produced complete hydrolysis and removalof the NHS chemistry, as well as removal of some of thethiolated OEG chains. NHS regeneration of the hydrolyzedsurfaces was assessed using the same multivariable peakintensity ratio as well as surface coupling with amine-terminated molecules. Both aqueous and organic NHSregeneration methods produced surfaces with bound NHSconcentrations ∼50% of the bound NHS concentration onfreshly prepared NHS-OEG monolayers. Precise methodsfor quantifying NHS chemistry on surfaces are usefulfor quality control processes required in surface technologies that rely on reliable and reproducible reactiveester coupling. These applications include microarray,microfluidic, immunoassay, bioreactor, tissue engineer-ing, and biomedical device fabrication.
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