Stefan Welin-Klintström

Monitoring specific interaction of low molecular weight biomolecules on oxidized porous silicon using ellipsometry

Porous silicon dioxide surfaces have been used for monitoring the specific affinity binding of low molecular weight molecules to streptavidin. Streptavidin was immobilized to the porous silicon dioxide surface by spontaneous adsorption at pH 7.4. Binding of biotin and an oligopeptide synthesized by means of combinatorial chemistry were monitored with an in situ null ellipsometer. Measurements were also done with hydroxy-azobenzene-2-carboxylic acid and DL-6-8-thioctic acid amide. The performance of porous silicon dioxide as a potential surface in biosensor applications was compared with a planar silicon dioxide surface. Porous silicon dioxide showed a 10-fold amplification of the response compared to planar silicon dioxide. It was possible to monitor the binding of biotin and the oligopeptide in the concentration range 2-40 microM. A response time as low as 30 s was obtained for the oligopeptide at 40 microM.

Competition between fibrinogen and a non-ionic surfactant in adsorption to a wettability gradient surface

The competition between mixtures of fibrinogen and a non-ionic surfactant (C12E5) with respect to adsorption onto a wettability gradient solid surface was studied by the use of ellipsometry. The effects of surface hydrophobicity and surfactant association were investigated. Furthermore the effect of clouding of the surfactant was studied by performing measurements at temperatures above and below the cloud point. At all concentrations, the fibrinogen (0.02–0.40 mg ml−1) was preferentially adsorbed onto the hydrophilic part of the gradient surface. At surfactant concentrations above and around the CMC, the protein was inhibited from adsorbing by the surfactant at the hydrophobic as well as in the intermediate part (50° ⩽ contact angle ⩽ 80°) of the gradient. As the surfactant concentrations was further reduced the protein was able to compete and adsorb onto the whole or parts of the gradient surface. In the case of a surfactant concentration of two-fifths of the CMC, the competitive power of the surfactant increased with temperature and the surfactant could hinder protein adsorption over a larger interval of the gradient surface. These observations were also verified by in situ measurements on non-gradient surfaces. The competition can be explained by considering the main interactions between protein and surfactant with the surface. In this respect cooperation in the self-association of the surfactant seems to be of great importance. The use of gradient surfaces makes it possible to observe subtle changes in these interactions.

The elutability of fibrinogen by sodium dodecyl sulphate and alkyltrimethylammonium bromides

Abstract The elutability of adsorbed fibrinogen by cationic surfactants of different chain lengths (dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, cetyltrimethylammonium bromide), and an anionic surfactant (sodium dodecyl sulphate (SDS)) was studied using in situ ellipsometry. The concentrations of the surfactants were twice the CMC in water and for fibrinogen, 0.4 mg ml−1. The investigation was carried out for two model surfaces: methylated silica (hydrophobic) and silica (hydrophilic and negatively charged, at pH 7). As a complement, a surface with a gradient in surface hydrophobicity was used. The end points of the gradient are similar to the methylated silica and silica surfaces with respect to hydrophobicity. All the surfactants adsorbed on the methylated silica surfaces, whereas only the cationic surfactants adsorbed on the silica surface. The adsorption of fibrinogen was 0.64 ± 0.03 μg cm−1 and 0.35 ± 0.03 μg cm−2 on the methylated silica and silica surfaces, respectively. Addition of surfactant led to a decrease in the amount of fibrinogen adsorbed on the methylated silica surface for all the surfactants, but only SDS affected the amounts adsorbed on the silica surfaces to any great extent. Despite the fact that the cationic surfactants adsorbed onto the silica surface, they did not affect the amount of fibrinogen adsorbed. The removal of protein decreased for the alkyltrimethylammonium bromides with increasing hydrophilicity of the gradient surfaces, and the amount of fibrinogen remaining after surfactant treatment decreased slightly for SDS. The effect of the chain length of the surfactant on elutability was small. The rate of removal of fibrinogen by the surfactants was found to be slower for SDS compared with the alkyltrimethylammonium bromides at the methylated silica surface, and at the hydrophobic end and in the intermediate part of the gradient. Adsorption from mixtures of surfactant and fibrinogen was also studied and the effects of cationic and anionic surfactants were quite different. The adsorption of fibrinogen was increased in the presence of the cationic surfactants, especially on the silica surface, but decreased in the presence of SDS. As surfactant adsorption onto clean surfaces is reversible with respect to dilution it might be assumed that the adsorbate mainly consists of fibrinogen. A trend was observed for the amounts of fibrinogen remaining after rinsing with buffer; the amounts increased with decreasing length of the surfactant hydrocarbon chain.

Comparison between wettability gradients made on gold and on Si/SiO2 substrates

We have previously shown that wettability gradients on Si:SiO2 can be used to analyze in detail the interactions of surfactants and proteins with the vast number of different surface properties that a gradient represents. We have also shown that the interactions of surfactants can be used to characterize the surface. In this report we discuss a new kind of wettability gradient that is composed from alkanethiols on gold. Surfactant and protein adsorption phenomena are investigated and compared with analogous experiments made on gradients prepared from chlorosilanes on Si:SiO2 substrates. Surfactant adsorption studies were used to investigate the surface properties. Through their electrostatic interactions negatively charged groups on the surface could be detected and through their hydrophobic interactions defects in the surface layer resulting in hydrophobic groups could be detected. The alkanethiol gradient had at the extreme OH-side an advancing contact angle to water between 30 and 40°. Still this surface had a lower adsorbed amount of fibrinogen compared to a (completely wetting) silica surface covered with negatively charged and uncharged silanol groups, indicating that the fibrinogen molecules also interacts electrostatically through their positively charged amino acid residues. This shows that the amount of fibrinogen adsorbed is not solely dependent on the surface hydrophilicity.

Nucleation as the rate-limiting step in the initial adsorption of ferritin at a hydrophobic surface

Abstract Adsorption of ferritin on a methyl silanized quartz surface was measured with in situ ellipsometry and transmission electron microscopy (TEM). An initial lag phase in the adsorption process followed by a rapid increase in the amount of bound ferritin was recorded by ellipsometry. The time dependence of the rapid adsorption exhibited an exponential form. The initial lag phase corresponds to a nucleation process manifested by the TEM studies. Adsorption was initiated at 109 sites per square centimetre, which corresponds well to the thickness measured by ellipsometry. The accelerated adsorption recorded by ellipsometry corresponds to an accelerating growth of protein clusters at the surface as seen by TEM. The present data indicate that the initial rate of adsorption of ferritin is limited by nucleation and not by diffusion. The exponential acceleration of nucleation and growth-like kinetics lead to depletion of protein in the reaction zone, which is the mechanism behind the often-seen mass transport limitation of macromolecular reactions at solid—liquid interfaces.

Molecular orientation of tridecafluorosilane on the surface of oxidized silicon

Abstract The orientations of tridecafluorosilane molecules adsorbed from solution onto Si(111) surfaces (with a thin oxide), were studied in ultra-high vacuum using angle-dependent X-ray photoelectron spectroscopy, XPS(θ). When adsorbed from dilute solutions, the adsorbed layer is very thin, since these linear molecules lie flat on the surface of the Si substrate. When adsorbed from concentrated solutions, however, the adsorbed layer is thicker, since the molecules exhibit a strong preference to stand up on the surface. The data on the thicker adsorbate layer are consistent with a molecular orientation where the major axis of the molecules makes an angle with the surface consistent with sp3 hybridization of the Si-atom in the silane molecule.

Proteolytic degradation of fibrinogen layers adsorbed on hydrophilic and hydrophobic surfaces

Abstract The adsorption of fibrinogen onto hydrophobic (methyl groups) or hydrophilic (hydroxyl groups) SiO 2 surfaces and the subsequent enzymatic degradation by trypsin was studied with the use of ellipsometry. The amount of adsorbed fibrinogen was 0.7 ng mm −2 on hydrophilic silicon and 3.0 ng mm −2 on hydrophilic silicon. On the hydrophilic surface 90% and on the hydrophobic surface 60% of the fibrinogen layer was degraded by trypsin. The effect of the wettability of the solid surface on the proteolytic degradation of the adsorbed fibrinogen layer was also studied with the use of a surface with graded wettability. A detection limit of ≈ 10 −4 mg ml −1 trypsin (corresponding to an enzymatic activity of ≈3.5·10 −4 u ml −1 ) at incubation times of 40 min was found. The undegradable 40% fibrinogen on the hydrophobic surface is discussed in relation to earlier findings. It was found with SDS-PAGE that the major part of this adsorbed layer had a molecular weight of ≈ 68 000.