X-ray Photoelectron Spectroscopy, Time-of-Flight Secondary Ion Mass Spectrometry, and Principal Component Analysis of the Hydrolysis, Regeneration, and Reactivity of N-Hydroxysuccinimide-Containing Organic Thin Films

Abstract

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. In this work, we use 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 multivariable 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. Precise methods for quantifying NHS chemistry on surfaces are useful for quality control processes required in surface technologies that rely on reliable and reproducible reactive ester coupling. These applications include microarray, microfluidic, immunoassay, bioreactor, tissue engineering, and biomedical device fabrication.

XPS, TOF-SIMS, NEXAFS, and SPR Characterization of Nitrilotriacetic Acid-Terminated Self-Assembled Monolayers for Controllable Immobilization of Proteins

“…In contrast, all OEG fragments emitted from the NTA thiol require breaking at least two bonds. Although there are situations where a multivariate peak ratio can properly track the surface concentration of a particular species (e.g., see reference 22 ), the results of the current study show it is always a good idea to examine the contributions of all peaks in the secondary ion spectra.…”

Covalently derivatized NTA microarrays on porous silicon for multi-mode detection of His-tagged proteins

“…The second should be related to the PEG brushes. The NHS-ester/amine linking reaction and hydrolysis of NHS-ester on surfaces have been investigated enormously for its importance on surface immobilization of biomolecules [34][35][36][37]. The incubation of the microarray chip in a saturated humidity within 1 hr will result in hydrolysis of NHS-ester less than 10% [37].…”

A comparative investigation of methods for protein immobilization on self-assembled monolayers using glutaraldehyde, carbodiimide, and anhydride reagents

“…However, the active ester is susceptible to hydrolysis. 31 In the present work, therefore, we used DMF as the solvent for the activation step. This was found to yield more reproducible data and also to yield better surface functionalization, irrespective of whether the subsequent immobilization step was aqueous ͑for proteins͒ or anhydrous ͑for amines in model studies͒.…”