Teodor Aastrup

Optimizing immobilization on two-dimensional carboxyl surface: pH dependence of antibody orientation and antigen binding capacity

The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two-dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We show that successful immobilization is highly dependent on surface pK(a), antibody pI, and pH of immobilization buffer. By the use of EDC/sulfo-NHS (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysulfosuccinimide) activation reagents, the effect of the intrinsic surface pK(a) is avoided and immobilization at very low pH is therefore possible, and this is important for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studied. In most cases, the antigen binding capacity followed the immobilization response. However, the antigen-to-antibody binding ratio differed between the antibodies investigated, and for one of the antibodies the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab(2) antibodies on different antibody surfaces indicated that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.

Experimental in situ studies of copper exposed to humidified air

Three complementary experimental techniques for in situ surface analysis have been combined for the first time in order to explore the chemistry and physics of a copper surface exposed to humidified air. Infrared reflection absorption spectroscopy, quartz crystal microbalance and atomic force microscopy provide a congruent picture of the processes occurring at the surface. At a given relative humidity, cuprous oxide forms according to an approximately logarithmic rate law. In addition, an aqueous adlayer of constant mass physisorbs on the surface. Increased relative humidity stimulates the physisorption of water and enhances the nucleation rate of oxide grains, thereby increasing the formation rate of cuprous oxide.

In Situ Studies of the Initial Atmospheric Corrosion of Copper Influence of Humidity, Sulfur Dioxide, Ozone, and Nitrogen Dioxide

Infrared reflection absorption spectroscopy and quartz crystal microbalance, integrated into a one-surface analytical system, and complemented with tapping mode atomic force microscopy, has been used to explore the metal/atmosphere interfacial region under atmospheric pressure conditions. This unique combination of in situ techniques, all possessing submonolayer sensitivity, has revealed information on the different accelerating roles of ozone (O 3 ) and nitrogen dioxide (NO 2 ) on the SO 2 -induced atmospheric corrosion of copper. The formation of reaction products could be followed quantitatively with respect to chemical identity and kinetics. Exposure in SO 2 -containing humidified air resulted in CuSO 3 .xH 2 O-like species, formed atop a cuprous oxide, designated Cu 2 O, all over the copper surface. O 3 introduction resulted in an accelerated mass gain with an increased formation rate of both Cu 2 O and of CuSO 4 .xH 2 O all over the surface. NO 2 introduction resulted in less mass gain than observed under SO 2 and O 3 , with no formation of new Cu 2 O, an initial oxidation of CuSO 3 .xH 2 O to CuSO 4 .xH 2 O, and with sulfite oxidation gradually replaced by copper nitrate formation, possibly as CuNO 3 (OH) 3 . The formation rates of the dominating end products, CuSO 4 .xH 2 O in SO 2 /O 3 and Cu 2 NO 3 (OH) 3 in SO 2 /NO 2 seemed to be limited by the supply of the gaseous constituents.

Photo-Click Immobilization on Quartz Crystal Microbalance Sensors for Selective Carbohydrate-Protein Interaction Analyses

A photoclick method based on azide photoligation and Cu-catalyzed azide-alkyne cycloaddition has been evaluated for the immobilization of carbohydrates to polymeric materials. The biomolecular recognition properties of the materials have been investigated with regard to applicable polymeric substrates and selectivity of protein binding. The method was used to functionalize a range of polymeric surfaces (polystyrene, polyacrylamide, poly(ethylene glycol), poly(2-ethyl-2-oxazoline), and polypropene) with various carbohydrate structures (based on α-D-mannose, β-D-galactose, and N-acetyl-β-D-glucosamine). The functionalized surfaces were evaluated in real-time studies of protein-carbohydrate interactions using a quartz crystal microbalance flow-through system with a series of different carbohydrate-binding proteins (lectins). The method proved to be robust and versatile, resulting in a range of efficient sensors showing high and predictable protein selectivities.

Multianalytical in situ investigation of the initial atmospheric corrosion of bronze

Atomic force microscopy (AFM) and infrared reflection absorption spectroscopy (IRAS) were used for in situ investigations of the initial atmospheric corrosion of bronze. In addition ex situ XPS inv ...

Photogenerated lectin sensors produced by thiol-ene/yne photo-click chemistry in aqueous solution.

The photoinitiated radical reactions between thiols and alkenes/alkynes (thiol-ene and thiol-yne chemistry) have been applied to a functionalization methodology to produce carbohydrate-presenting surfaces for analyses of biomolecular interactions. Polymer-coated quartz surfaces were functionalized with alkenes or alkynes in a straightforward photochemical procedure utilizing perfluorophenylazide (PFPA) chemistry. The alkene/alkyne surfaces were subsequently allowed to react with carbohydrate thiols in water under UV-irradiation. The reaction can be carried out in a drop of water directly on the surface without photoinitiator, and any disulfide side products were easily washed away after the functionalization process. The resulting carbohydrate-presenting surfaces were evaluated in real-time studies of protein-carbohydrate interactions using a quartz crystal microbalance (QCM) flow-through system with recurring injections of selected lectins, with intermediate regeneration steps using low pH buffer. The resulting methodology proved fast, efficient and scalable to high-throughput analysis formats, and the produced surfaces showed significant protein binding with expected selectivities of the lectins used in the study.

Real-time analysis of the carbohydrates on cell surfaces using a QCM biosensor: a lectin-based approach

A novel approach to the study of molecular interactions on the surface of mammalian cells using a QCM biosensor was developed. For this study, an epidermoid carcinoma cell line (A-431) and a breast adenocarcinoma cell line (MDA-MB-468) were immobilized onto polystyrene-coated quartz crystals. The binding and dissociation between the lectin Con A and the cells as well as the inhibition of the binding by monosaccharides were monitored in real time and provided an insight into the complex avidic recognition of cell glycoconjugates. The real-time lectin screening of a range of lectins, including Con A, DBA, PNA and UEA-I, enabled the accurate study of the glycosylation changes between cells, such as changes associated with cancer progression and development. Furthermore, the kinetic parameters of the interaction of Con A with MDA-MB-468 cells were studied. This application provides investigators in the field of glycobiology with a novel tool to study cell surface glycosylation and may also have impacts on drug discovery.

Combined in-situ investigations of atmospheric corrosion of copper with SFM and IRAS coupled with QCM

Abstract Scanning force microscopy (SFM) and infrared reflection absorption spectroscopy (IRAS) coupled with quartz crystal microbalance (QCM) were used for in-situ investigations of the atmospheric corrosion of pure copper. Based on chemical and kinetic studies obtained by IRAS/QCM, tapping mode atomic force microscopy (TM-AFM) and phase detection imaging (PDI) were applied to gain information about the topography changes of the sample surface with emphasis on the shape and lateral distribution of the corrosion products. Investigations were carried out in synthetic air with 60 and 80% relative humidity (RH). At 60% RH, small features partly covering the surface could be observed with TM-AFM. These features were identified as cuprous oxide with IRAS. Contrary to these results, a fast formation of a layer of cuprous oxide entirely covering the sample surface was observed at 80% RH with TM-AFM. QCM investigations showed a higher formation rate of cuprous oxide at 80% than at 60% RH but in both cases, a high corrosion rate at the beginning of the exposure, which decreases with progressing time.

Surface-confined photopolymerization of pH-responsive acrylamide/acrylate brushes on polymer thin films

Dynamic acrylamide/acrylate polymeric brushes were synthesized at gold-plated quartz crystal surfaces. The crystals were initially coated with polystyrene-type thin films, derivatized with photolabile iniferter groups, and subsequently subjected to photoinitiated polymerization in acrylamide/acrylate monomer feeds. This surface-confined polymerization method enabled direct photocontrol over the polymerization, as followed by increased frequency responses of the crystal oscillations in a quartz crystal microbalance (QCM). The produced polymer layers were also found to be highly sensitive to external acid/base stimuli. Large oscillation frequency shifts were detected when the brushes were exposed to buffer solutions of different pH. The dynamic behavior of the resulting polymeric brushes was evaluated, and the extent of expansion and contraction of the films was monitored by the QCM setup in situ in real time. The resulting responses were rapid, and the effects were fully reversible. Low pH resulted in full contractions of the films, whereas higher pH yielded maximal expansion in order to minimize repulsion around the charged acrylate centers. The surfaces also proved to be very robust because the responsiveness was reproducible over many cycles of repeated expansion and contraction. Using ellipsometry, copolymer layers were estimated to be approximately 220 nm in a collapsed state and approximately 340 nm in the expanded state, effectively increasing the thickness of the film by 55%.

A comparison of preparation methods of copper surfaces for in situ scanning force microscopy investigations

Scanning force microscopy (SFM) studies of atmospheric corrosion of pure copper require a clean, homogeneous and smooth metal surface in order to image small changes. In this study various preparation methods for samples of copper sheets were tested: chemical etching, mechanical polishing as well as electrochemical polishing. In addition to copper sheet samples also specimen of sputtered copper obtained by physical vapour deposition on quartz substrates were chemically etched. In conclusion, mechanical polishing with monocrystalline diamond paste and electrochemical polishing of copper sheet yielded the most suitable surface condition for in situ investigations by means of SFM. Tapping mode atomic force microscopy (TM-AFM) was applied to gain information about the topography changes of the copper surface. Furthermore, the root mean square (rms) roughness of the sample surfaces was determined and delivered additional arguments for the applicability of the various surface treatments for in situ studies.