Spectroscopic Examination of Protein Adsorption from Seawater onto Titanium

Abstract

Several noninvasive optical and electrochemical techniques were adapted to examine partitioning of protein from seawater onto polished titanium with the use of the plant enzyme ribulose-1,5,-bisphosphate carboxylase-oxygenase (Rubisco) as a model. Protein films, varying in surface concentrations from 0.011 to 3.606 μg cm−2, were prepared by exposing polished Ti surfaces to seawater amended with 0.04–90.80 μg mL−1 of 3H-Rubisco. Mean film thickness, d, measured by ellipsometry, increased linearly over most of the range of irreversibly bound protein (Γirr = 0.011–2.491 μg cm−2). Spatial coverages of the films were more heterogeneous at low surface coverages, indicative of heterogeneous adsorption resulting in barren Ti oxide surface sites and insular protein clusters. The thickness of the underlying Ti oxide layer, also measured by ellipsometry, was highly variable and indicated that oxidation of the surface was suppressed at high protein coverages during two-hour exposures to seawater. Vibrational spectra of surface films, from submonolayer (0.03 μg cm−2) to multilayer (3.61 μg cm−2), were obtained with the use of Fourier transform infrared reflection-absorption spectrometry (FT-IRAS). Peak areas of amide I and II bands varied linearly with Γirr, permitting noninvasive measurement of protein mass at the surface. Relative intensities of the amide II/amide I bands, band composition of the amide III, and peak frequencies varied with surface concentration, indicating unfolding of adsorbed proteins. Vibrational spectroscopic and ellipsometric evidence suggests that protein structure is most altered at low surface concentrations. Electrochemical impedance spectroscopy (EIS) performed from 100 /μHZ to 100 kHz on replicate test surfaces revealed that the electrochemical behavior of the titanium/protein interface was consistent with that of a parallel RC circuit. The charge transfer resistance, Rct, of the interface varied as a two-state function of protein concentration. The Rct increased more rapidly within the monolayer domain (0.12 to 2.8 MΩ cm2) than in the multilayer domain (2.8 to 4.9 MΩ cm2), indicating that impedance to electron flow across the interface is most influenced by protein monolayer formation and is less affected by additional layers. Estimations of rates of oxidation or dissolution of the substratum were inversely proportional to protein surface concentrations. Together these techniques provide internally consistent measurements of surface film thickness, adsorbate mass, gross chemical composition, interface organization, electrical impedance, capacitance, and oxide layer thickness. These data are useful for determining the physical state of the interface, its dynamics, and the potential oxidation rates of the substratum underlying the surface film.

Biomaterial Interface Investigated by Electrochemical Impedance Spectroscopy

“…3(c)), which further increases with higher protein concentration. [46] The CTR (R 2 in Fig. 3(o)) decreases with increasing protein concentration and increasing potential.…”

“…[40] Figure 3(c) shows a simple circuit with sufficient fitting results, which is used very often. [4,12,26,29,32,36,[42][43][44][45][46][47][48] As a rule this equivalent circuit is used for a metal in contact with an electrolyte and it consists of the resistance of the solution R sol , a capacitance and the resistance R 1 , which can be a CTR [4,12,32] or the resistance of the passive layer. [32] The CTR represents the limited charge transfer through the interface and the electric double-layer capacitance.…”

Enantioselective Interactions at the Solid–Liquid Interface of an HPLC Column under Working Conditions

“…Recently, the feasibility of using an ATR-IR microscope as a detection unit has been also demonstrated . Several coating materials like polymers, – biological materials, – and sol–gel films , have been used to increase the detection limits of the ATR-IR technique for quantification. The coating has the advantage to increase the population density of molecules nearby the internal reflection element (IRE) used as waveguide for the infrared radiation.…”