Coadsorption-dependent orientation of fibronectin epitopes at hydrophilic gold surfaces
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The effect of coadsorption on the conformational arrangement of human plasma fibronectin (Fn) was studied for mixtures with human serum albumin (HSA) adsorbed onto mildly hydrophilic gold substrates. Quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM) were used to measure the mass uptake, thickness, viscoelastic behaviour, and morphology of the adsorbed protein adlayers. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was employed to determine the composition of binary protein adlayers, taking advantage of the principal component analysis (PCA) technique of ToF-SIMS data. Thus, the ToF-SIMS results provided the particular fragmentation patterns of the two proteins, showing that the resulting mixed protein layers were predominantly formed by Fn molecules, even for binary solutions with high molar fraction of HSA. The conformational arrangement of the Fn molecules was studied by combining ToF-SIMS and QCM-D techniques. ToF-SIMS data allowed the identification of Type I–Type III modules of Fn and showed that pure Fn layers predominantly expose Type III modules, while coadsorbed Fn/HSA layers predominantly expose Fn Type I epitopes. QCM-D was employed to measure the relative uptake of a polyclonal antibody (anti-Fn) to the 4F15F1 binding domain in the Fn Hep I fragment in Type I modules, showing that pure Fn adlayers have a reduced anti-Fn binding capacity, as expected for Type I modules buried within the adlayers, while coadsorbed Fn layers bind more efficiently the anti-Fn, as the concerned Type I module is predominantly exposed at the layer surface. The results overall demonstrated that coadsorption of Fn and HSA onto mildly hydrophilic gold substrates prompts Fn to undergo a closed-to-open conformational switch.Keywords:
Quartz Crystal Microbalance
Human serum albumin
Auger electron spectroscopy
Electron spectroscopy
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Two different phosphonic acid monolayer films for immobilization of bioactive molecules such as the protein BMP-2 on titanium surfaces have been prepared. Monolayers of (11-hydroxyundecyl)phosphonic acid and (12-carboxydodecyl)phosphonic acid molecules were produced by a simple dipping process (the T-BAG method). The terminal functional groups on these monolayers were activated (carbonyldiimidazole for hydroxyl groups and N-hydroxysuccinimide for carboxyl groups) to bind amine-containing molecules. The reactivity of the surfaces was investigated using trifluoroethylamine hydrochloride and BMP-2. Each step of the surface modification procedure was characterized by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry.
Self-assembled monolayer
Surface Modification
Reactivity
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Fourier transform spectroscopy
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The sensitivity and the dynamic range of a quadrupole based mass spectrometer for the quasisimultaneous analysis of secondary ions and electron beam postionized sputtered neutrals has been improved. Counting rates larger than 106 counts/s of postionized neutrals with a dynamic range of six orders of magnitude have been obtained for iron samples bombarded by 10-μA, Ar+, 5 keV. A comparison of secondary ion mass spectrometry (SIMS) and secondary neutral mass spectroscopy (SNMS) results considering target preparation, sensitivity, reproducibility, accuracy of quantification, and total time of analysis will be presented for 13 steel targets alloyed with up to 15 different elements. Concentration ranges between 8 ppm and 1% have been covered. From the experimental results we found that the detection limit is in the range of 10−4 (oxygen) of a monolayer on a target area of 4 mm2, when the spectrometer is tuned to a distinct mass. This allows quantitative surface and interface analysis in a concentration range below Auger electron spectroscopy.
Auger electron spectroscopy
Secondary electrons
Quadrupole mass analyzer
Hybrid mass spectrometer
Secondary emission
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Using the quartz crystal microbalance (QCM) technique, we demonstrate that the contribution of Hg adsorption and absorption on the sensor response profile can be distinguished by studying the dynamic response curve of QCM based Hg vapor sensors that employ an ultra-thin film of Au in the range of 10 to 40 nm thickness as the sensitive layer. The response magnitudes of the QCMs were extrapolated to zero thickness (ETZT) in an attempt to determine the contribution of adsorbed Hg on the sensor response magnitude and response profile. In general, the ratio of adsorbed to absorbed Hg on Au films is found to decrease with increased Hg vapor concentration. Furthermore, the same ratio was observed to decrease with increasing Au film thickness. The 10 nm and 40 nm Au films for example were found to contain adsorbed Hg content of 43.8% and 16.4%, respectively, with the balance attributed to absorption/amalgamation, when exposed to Hg vapor concentration of 10.55 mg m−3 for a period of 14 hours and an operating temperature of 28 °C. In addition, the QCMs were characterized using secondary ion mass spectroscopy depth profiling in order to study the diffusion behaviour of Hg in the Au surfaces. It is deduced that in order to reduce Hg accumulation in Au thin films, a non-continuous type film (similar to the 10 nm ultra-thin Au sensitive layer morphology) would be more functional as a Hg sensitive layer where quick absorption and desorption processes are required.
Quartz Crystal Microbalance
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Chemisorption
Noble gas
Density of states
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