Real Time Evaluation of Composition and Structure of Concanavalin A Adsorbed on a Polystyrene Surface

Abstract

In situ qualitative and quantitative evaluations of adsorbed submonolayers and multilayers of the protein Concanavalin A (ConA) on a polystyrene surface were carried out by attenuated total reflection infrared spectroscopy. The influence of pH and adsorption time on the composition and structure of the adsorbed protein layers was investigated by comparison of the experimental spectra with simulated spectra of hypothetical multilayer systems with the assumed composition, thickness, and structure. This methodology allows the differentiation of observed spectral changes that result from pure optical effects, associated with the passing of an incident beam through the multilayer system, from the chemical and structural changes caused by physicochemical interactions of proteins with polymer surfaces. This represents significant progress since small variations in the band positions or intensities of amide I and amide II infrared absorbance bands have an important interpretation consequence. The applied methodology significantly reduces the misinterpretation of recorded spectra of protein layers and is rewarded by a deep insight of the structure and composition of the samples. The composition, structure, and kinetics of the adsorption of ConA and hydration level of the adsorbed layers were evaluated in detail. Competitive adsorption of bovine serum albumin on pre-adsorbed ConA layers also was investigated to characterize the ConA surface distribution. Parallel studies using X-ray photoelectron spectroscopy support the conclusions drawn from infrared spectroscopic investigations on ConA molecular distributions at the polymer surface. Two-step models that describe ConA submonolayer formation at pH 4.8 and multilayer formation at pH 7.8 are proposed.

Efficiency of blocking of non-specific interaction of different proteins by BSA adsorbed on hydrophobic and hydrophilic surfaces

“…However, the experimental spectra can be interpreted accurately by generating a simulated spectrum for the adsorbed protein layers with assumed thickness and composition using the solid state (native) optical constants of the proteins, which account for all optical effects [23]. Evaluation of the thickness of the adsorbed protein layers by spectral simulation enables the surface area covered by the adsorbed protein molecules to be determined quantitatively and precisely [23,24]. The simulated absorbance spectra of the proteins were calculated from Hansen's equations [25].…”

“…The simulated absorbance spectra of the proteins were calculated from Hansen's equations [25]. The optical constants of the layers, such as those of PS, BSA, Con A, IgG and SpA, were taken from our previous studies [23,24]. A stratified multilayer model with a homogeneous layer for each component phase (as shown in Fig.…”

“…A stratified multilayer model with a homogeneous layer for each component phase (as shown in Fig. 1) was adopted for the spectral simulation, as described in detail in our previous studies [23,24].…”

Protein Adsorption on a Glycosylated Polyacrylonitrile Surface: Monitoring with QCM and SPR

“…Con A in the form of tetramer at pH ¼ 8.5 is a hydrophilic protein (M % 10 5 , dimension 67 Â 113 Â 122 Å 3 [41] ), which is strongly hydrated with about 50 wt.-% water. [42] Therefore, the factor of 2.0 is used to estimate the resonant frequency shift in dry form. Based on Sauerbrey's equation, the loading mass of 1.58 mg Á cm À2 was calculated from the resonant frequency shifts 270 Hz in Protein Adsorption on a Glycosylated Polyacrylonitrile .…”

Modification of polystyrene surface in aqueous solutions

“…The drawing speed was controlled at 18 mm/min. PS films that were $16-nm thick were obtained from 10 mg/mL coating solution of PS, as described previously [52,53].…”