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

Abstract

For immobilization of proteins onto surfaces in a specific and controlled manner it is important to start with a well-defined surface that contains specific binding sites surrounded by a nonfouling background. For immobilizing histidine-tagged (his-tagged) proteins, surfaces containing nitrilotriacetic acid (NTA) headgroups and oligo(ethylene glycol) (OEG) moieties are a widely used model system. The surface composition, structure and reactivity of mixed NTA/OEG selfassembled monolayers (SAMs) on Au substrates were characterized in detail using x-ray photoelectron spectroscopy (XPS), near-edge x-ray absorption fine structure spectroscopy (NEXAFS), time-of-flight secondary ion mass spectrometry (ToF-SIMS) and surface plasmon resonance (SPR) biosensoring. XPS results for sequential adsorption of NTA thiols followed by OEG thiols showed that OEG molecules were incorporated into an incompletely formed NTA monolayer until a complete mixed SAM was formed. The surface concentration of NTA headgroups was estimated to be 0.9-1.3 molecule/nm 2 in the mixed NTA/OEG monolayers, compared to 1.9 molecule/nm 2 in pure NTA monolayers. Angle-dependent XPS indicated NTA headgroups were slightly reoriented toward an upright position after OEG incorporation and polarization-dependent NEXAFS results indicated increased ordering of the alkane chains of the molecules. Nitrogen-containing and OEG-related secondary ion fragments from the ToF-SIMS experiments confirmed the presence of NTA headgroups and OEG moieties in the monolayers. A multivariate peak intensity ratio was developed for estimating the relative NTA concentration in the outermost (10 Ǻ) of the monolayers. SPR measurements of a his-tagged, humanized antilysozyme variable fragment (HuLys Fv) immobilized onto Ni(II)-treated mixed NTA/OEG and pure NTA monolayers demonstrated the reversible, site-specific immobilization of his-tagged HuLys Fv (108 -205 ng/cm 2 ) with dissociation rates (k off ) between 1.0x10 −4 and 2.1x10 −5 s −1 , both depending on the NTA surface concentration and orientation. The monolayers without Ni(II) treatment exhibited low nonspecific adsorption of his-tagged HuLys Fv ( < 2ng/cm 2 ).

Immobilization of His-tagged kinase JAK2 onto the surface of a plasmon resonance gold disc modified with different copper (II) complexes

“…In the literature, various methods for NTA molecule immobilization on the surface of the SPR discs were described and the binding constants for these systems are very similar to the ones described by Nieba et al (~1.0x10 6 M -1 ) [20,[41][42][43]. Nakamura et al have studied different anchors for immobilization of NTA molecules onto the gold surface which were chelated with nickel(II) [25].…”

An Organic Transistor-based Electrical Assay for Copper(II) in Water

“…Importantly, the peak values of binding energy in each region (C1s, N1s, O1s, and S2p) are almost identical with past reports for the characterization of the NTA-SAM on the Au film. 21 Moreover, the CAG was carried out to estimate the surface tension of the Au film modified with the NTA-SAM. When the surface tension (£ i ) (i = S; solid or L; liquid) is assuming that to be the sum of a dispersion component (£ i d ) and a polar component (£ i p ), 22 the relationship between the contact angle (ª) of a liquid on a solid surface and the surface tension of the solid surface (£ S ) can be described as the following Eq.…”

Analysis of the surface density and reactivity of perfluorophenylazide and the impact on ligand immobilization

“…This resonance is commonly related to the p* resonance from C¼C bonds. [32][33][34][35][36][37][38] However, upon fluorination, the p* C¼C resonance is shifted by as much as 5 eV, 21,37 and therefore, the peak at 289.2 eV (labeled 4) is also related with the F-C*¼C-F p* resonance. In addition, the two PFPA-MUTEG/MDEG mixed films present a shoulder at 288.2 eV (labeled 3) and a peak at 289.8 (labeled 5).…”