Hans Elwing

Structure of 3-aminopropyl triethoxy silane on silicon oxide

Abstract 3-Aminopropyl triethoxy silane (APTES) was deposited onto silicon oxide surfaces under various conditions of solvent, heat, and time, and then exposed to different curing environments, including air, heat, and ethanol. The macro- and micromolecular structure of APTES was probed on different levels using different techniques. The thickness of APTES layers was measured by ellipsometry, macromolecular structures were identified using microscopic methods (scanning electron microscopy and atomic force microscopy), and chemical form was investigated using angle-resolved X-ray photoelectron spectroscopy and also reflection infrared spectroscopy of APTES on gold and aluminum oxide surfaces. Coverage equivalent to one monolayer was achieved using very mild reaction and curing conditions (reaction in dry toluene for 15 min at room temperature, curing in air, or 15 min in 200°C oven), whereas thick layers were made by increasing reaction and curing times. APTES initially adsorbed to the surface, and curing was necessary to complete covalent binding between APTES and the surface. Deposition of APTES from water gave thin layers, probably electrostatically bound to silicon.

Conformational changes of a model protein (complement factor 3) adsorbed on hydrophilic and hydrophobic solid surfaces

Abstract We have used hydrophobic and hydrophilic silicon surfaces as model surfaces to study the importance of solid surface wettability for the conformation of adsorbed C3 molecules. Adsorption of C3 and its subsequent interaction with antibodies were measured by enzyme-linked immunosorbent assay and ellipsometry. Immunochemical evidence for conformational changes of the adsorbed C3 molecule was investigated by the use of antibodies directed against epitopes hidden in the native molecule. It was found that C3 adsorbed on hydrophobic silicon had a conformation exposing antigenic epitopes which are only accessible in C3 denatured by SDS or C3 that have been biologically activated.

Structure of adsorbed fibrinogen obtained by scanning force microscopy

It is shown that scanning force microscopy (SFM), operated in the attractive mode, can be used to obtain high resolution pictures of adsorbed fibrinogen molecules on solid surfaces, without the need for staining or special microscope grids. SFM also reveals the three‐dimensional structure of the adsorbed molecules. Two forms of adsorbed fibrinogen are demonstrated on hydrophobic silicone dioxide surfaces; a trinodular about 60 nm long and a globular with about a 40 nm diameter. Polymeric networks formed after storage of the surface with adsorbed fibrinogen in PBS for 11 days are also shown. The SFM‐results for the trinodular structure suggest the existence of loops or peptide chains extending outside the basic structure of the fibrinogen molecule.

Protein immobilization of 3-aminopropyl triethoxy silaneglutaraldehyde surfaces: Characterization by detergent washing

Abstract With the use of sodium dodecyl sulfate, covalent binding of protein was distinguished from nonspecific adsorption to 3-aminopropyl triethoxy silane (APTES) glutaraldehyde-treated surfaces. In this experiment, APTES was immobilized i a concentration gradient on silicon dioxide wafers, reacted with glutaraldehyde, and then incubated with albumin, fibrinogen, or immunoglobulin G. The so-formed gradients contained both experimental and control surfaces on extreme ends of the wafer, with varying proportions of the two in the middle. Each reaction step was examined by ellipsometry, a sensitive optical method for quantifying thin layers. This “gradient technique” substantially reduced the workload and simplified interpretation of the results. It was found that nonspecific adsorption of protein, especially to hydrophobic control surfaces, was greatly reduced when Tween 80 (a nonionic detergent) was added to the protein incubation solution. With the use of liquid/solid ellipsometry, it was found that the majority of glutaraldehyde and of immunoglobulin G adsorbed to the surface in the first 5 min.

Desorption of fibrinogen and γ-globulin from solid surfaces induced by a nonionic detergent

Abstract The action of a nonionic detergent (Tween 20) on adsorbed layers of fibrinogen or γ-globulin on solid surfaces was studied. The solid surface used was a hydrophilic silicon dioxide surface with a gradient of hydrophobic methyl groups (wettability gradient surface). The amount of adsorbed organic material on the gradient surface was determined with the use of an optical method (ellipsometry) with sufficiently high lateral resolution. It was demonstrated that Tween 20 has a small effect on desorption of adsorbed proteins on the hydrophilic side of the gradient. Desorption of proteins occurred, however, at the hydrophobic side of the gradient. Desorption of the protein layers was strongly inhibited by incubation of the adsorbed protein layers in buffer at 37°C for 4 h before incubation in Tween 20. A probable explanation for this is that the adsorbed protein molecules gradually increase their hydrophobic interaction with the surface during the incubation time.

Reflectometry in kinetic studies of immunological and enzymatic reactions on solid surfaces

Abstract An optical method is described, by means of which immunological and enzymatic reactions can be followed at a primary level on a solid surface, without labelling procedures. When plane-polarized light is reflected at a solid surface, there is a minimum in reflectance at a certain angle of incidence, the pseudo-Brewster angle. For example, a layer of protein adsorbed on a silicon surface increases the reflectance with increasing amount of adsorbed material. High sensitivity is obtained because of the large difference in refractive index between silicon and organic material; about 0.1 μg cm −2 adsorbed protein can be detected. In a model system of human IgG and anti-human IgG, the primary adsorption of IgG on a hydrophobic surface is first measured, and on this IgG-coated surface the binding kinetics of anti-IgG could be measured. The kinetics of proteolytic degradation of IgG-coated surfaces by trypsin was also investigated.

Determination of proteolytic activity: A sensitive and simple assay utilizing substrate adsorbed to a plastic surface and radial diffusion in gel

Abstract A method to determine proteolytic activity using radial diffusion in agar gel in combination with protein adsorbed to a polystyrene surface as substrate is developed. After the agar gel is removed, the effect of enzyme activity is demonstrated by condensation of water vapor on the protein-coated surface. Wettability of the enzyme-affected surface is decreased compared to that of the unaffected protein surface. The method is tested with two enzymes, trypsin and pronase. The sensitivity of the method is on a par with other highly sensitive methods. With bovine serum albumin (BSA) as substrate, the activity in 1 ng of enzyme is detected in a 10 μl test volume. A linear relationship is found between the enzyme-affected surface and the logarithmic value of the enzyme concentration from 0.1 μg/ml to 1 mg/ml. With 5 μg/ml BSA in distilled water, the polystyrene surface is sufficiently coated for use in the assay. The method also permits quantitative protease determination in crude and contaminated samples. This sensitive and simple technique has a high degree of accuracy and reproducibility without several of the disadvantages associated with other types of methods.

Fish scales as biosensors

Abstract It is demonstrated how certain fish scales (in our case from Cuckoo Wrasse, Labrus ossifagus) can be used for biosensing purposes. These scales contain cells with pigment granules which are either dispersed or aggregated. The pigments of an isolated fish scale are, e.g., aggregated by noradrenaline or other catecholamines. The aggregation can be measured by a simple photometer or by fibre optical probes. The sensitivity is very large. Nanomoles of noradrenaline can be easily measured. We discuss therefore the possibility of developing sensor probes for clinical use based on the fish scales.

'Lens-on-surface' : a versatile method for the investigation of plasma protein exchange reactions on solid surfaces

The exchange sequence of plasma proteins in narrow spaces on solid surfaces was studied by means of a modified lens-on-surface method as originally described by Vroman and Adams. In our studies, lateral scanning ellipsometry was used as the detection method. With the use of antibodies it was demonstrated and confirmed that immunologically detectable plasma protein antigens appear and disappear in a time- and concentration-dependent sequence [IgG followed by fibrinogen followed by high-molecular weight kininogen (HMWK)] on silica surfaces. Plasma protein exchange reactions were also studied on hydrophilic titanium (Ti), vanadium (V), and silver (Ag) surfaces. Atypical exchange patterns were found on V and Ag surfaces as compared with hydrophilic silica (adsorbed fibrinogen was not removed).

Proteins adsorbed to a hydrophobic surface used for determination of proteolytic activity

Abstract Wettability of a thin layer of protein adsorbed to a hydrophobic surface is reduced after proteolytic digestion. Reduced wettability is demonstrated by condensation of water vapour on the surface. The condensation patterns of enzyme-treated and untreated protein layers give different light-scattering properties which can be observed by the naked eye. Based on these principles, a new simple and inexpensive method, thin layer enzyme assay (TEA), for determination of proteolytic activity, was developed. Fibrinogen, gammaglobulin (IgG), bovine serum albumin (BSA), haemoglobin, ovalbumin and gelatin were used as substrates. The proteolytic activity in 1 ng trypsin (EC 3.4.21.4) and in 1 ng pronase (EC 3.4.24.4) was reproducibly detected.