Surface Characterization of Mixed Self-Assembled Monolayers Designed for Streptavidin Immobilization

Abstract

The self-assembly of streptavidin onto biotinylated alkylthiolate monolayers on gold has served as an important model system for protein immobilization at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled monolayers (SAMs) containing biotinylated and diluent alkylthiolates and their use to specifically immobilize streptavidin. X-ray photoelectron spectroscopy (XPS), angle-resolved XPS (ARXPS), near-edge X-ray absorption fine structure (NEXAFS), and surface plasmon resonance (SPR) have been used to characterize the films produced on gold from a range of binary mixtures of a biotinylated alkylthiol (BAT) and either a C16 methyl-terminated thiol (mercaptohexadecane, MHD) or a C11-oligo(ethylene glycol)-terminated (OEG) thiol in ethanol. The correlation between the solution mole fraction of BAT and its surface mole fraction (χBAT,sur) indicates that it adsorbs ∼4-fold faster than OEG but slightly slower than MHD. ARXPS analysis demonstrates that the biotin terminus of the BAT is exposed at the surface of mixed monolayers with χBAT,sur < 0.5 but is randomly distributed through BAT-rich films. Thus, the OEG diluent not only adds nonfouling properties but induces an improved concentration of biotin at the surface and reduces the exposure of the methylene segments of BAT. NEXAFS characterization demonstrates that pure OEG and mixed BAT/OEG SAMs do not show significant anisotropy in C−C bond orientation, in contrast to MHD and mixed BAT/MHD SAMs, whose aliphatic segments exhibit pseudo-crystalline packing. SPR measurements of streptavidin binding to and competitive dissociation from the different mixed SAMs indicate that streptavidin binds both specifically and nonspecifically to the BAT/MHD SAMs but purely specifically to BAT/OEG SAMs with χBAT,sur < 0.5. For BAT/OEG mixtures with χBAT,sur = 0.1−0.5, specifically bound streptavidin coverages of ∼80% of the C(2,2,2) two-dimensional streptavidin crystalline density (∼280 ng/cm2) can be reproducibly achieved. These composite results clarify the relationship between the specificity of streptavidin recognition and the surface architecture and properties of the mixed SAMs.

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

“…Since the thiol groups are located at the gold-monolayer interface, the lower sulfur concentrations are due to the attenuation of sulfur photoelectrons by overlying organic layers. 15,18,47 NTA a and NTA b were exposed to OEG diluent thiol for 0.5 hr to 48 hr. Composition trends observed suggests that the OEG thiol diluent produced different results for NTA a and NTA b.…”

“…49 After the addition of OEG diluent thiol, spectra show the presence of unbound sulfur in the 163 to 165 eV range, possibly due to incomplete rinsing of the sample surface or retention of physically adsorbed thiols. 15,49 Surface concentration of NTA headgroups in pure NTA and mixed NTA/OEG monolayers-The surface concentration of the NTA thiol is estimated using the method published by Nelson et al 15 and the results are summarized in Table 1. Applying this method to the measured XPS compositions, we assume the molecular volume for NTA and OEG in the mixed monolayers was the same as the values in their respective pure monolayers.…”

“…This parameter for the OEG molecules is consistent with the previously reported values. 15 Since nitrogen is unique to the NTA thiol, the nitrogen content is used to calculate surface concentration of NTA headgroups in the mixed NTA/OEG monolayers. The NTA concentration in the NTA b monolayer is calculated by the degree of attenuation of the substrate Au 4f XPS signal.…”

“…[40][41][42][43][44] The calculation of the protein surface coverage from the SPR response is described elsewhere. 15,16 The home-built SPR liquid biosensing system used in this study has been described and characterized in more detail elsewhere. 45,46 A dual-channel Teflon flow cell with two independent, low volume parallel flow channels was used.…”

“…One attractive way to anchor proteins involves preparing a surface of dispersed bioactive ligands (e.g. biotin 15,16 ) in a protein resistant background (e.g. oligo(ethylene glycol) [17][18][19] ).…”