XPS, TOF-SIMS, NEXAFS, and SPR Characterization of Nitrilotriacetic Acid-Terminated Self-Assembled Monloyers for Controllable Immobiliztion of Proteins
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 self-assembled 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/nm2 in the mixed NTA/OEG monolayers, compared to 1.9 molecule/nm2 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 Angstroms ) of the monolayers. SPR measurements of a his-tagged, humanized anti-lysozyme 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/cm2) with dissociation rates (koff) between 1.0 x 10-4 and 2.1 x 10-5 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 (<2 ng/cm2).
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 959534
- Report Number(s):
- BNL-82520-2009-JA; TRN: US1005754
- Journal Information:
- Analytical Chemistry, Vol. 80
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS
99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE
ABSORPTION
ADSORPTION
ALKANES
CHAINS
DISSOCIATION
FINE STRUCTURE
MASS SPECTROSCOPY
NTA
ORIENTATION
PLASMONS
PROTEINS
RESONANCE
SPECTROSCOPY
SUBSTRATES
THIOLS
X-RAY PHOTOELECTRON SPECTROSCOPY
national synchrotron light source