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Acoustic wave based immunosensors have proven to facilitate the in situ detection of marker-free proteins in real time. However, the vast majority of these studies focuses on the interaction of a single type of antigen with immobilized receptors in pure buffer solutions. In an effort to evaluate the potential of acoustic plate mode immunosensors for operation in more complex biological environments, antigen/antibody reactions have been studied in pure buffer solution, in the presence of cells, and in human serum. It has been observed that the devices do not respond to cell adsorption and that antigen/antibody reactions can successfully be detected even if a thick layer of cells is deposited on the sensing surface. By varying the frequency of operation, it was shown that the sensitivity of the devices toward nonspecific protein adsorption is reduced at high frequencies of operation. Thus, spurious immunosensor response caused by nonspecific adsorption processes can be suppressed by appropriately selecting device frequency. Using immunoglobulin G with minimum cross reactivity with human serum proteins, antigen/antibody reactions have also been monitored in human serum. While the observed frequency shifts are comparable to those measured in pure buffer solutions, the binding process is accompanied by additional acoustic loss, indicating changes in the viscoelastic properties of the interfacial layer.

A practical immunusensor, based on a 150-MHz acoustic plate mode (APM) device on ZX-LiNb[O.sub.3] has been built and is employed to monitor directly, in real time, antigen-antibody reactions via mass loading of the crystal surface. The results show frequency shifts [approximately] 1 kHz above the noise level upon antigen adsorption. The mass density detection limit of our present sensor is [approximately] 200 pg/m[m.sup.2], corresponding to [8.10.sup.8] molecules/m[m.sup.2] utilizing a typical IgG of 150 000 Da. Antigen binding capacities of aminosilane films are measured independently by a modified ELISA test and are related to the APM sensor response.

Protein unfolding and loss of proteinfunction upon surface contactis a major problem in biotechnology and biomedicine. Using glucoseoxidase (GOx) as a model protein, we investigated the impact of surfacechemistry, topography, and confinement on enzyme activity, conformation,and affinity. A particular focus lay on the question whether the conformationof surface-bound proteins can be stabilized by embedding nanoscaleadsorption sites, here in the form of monodisperse gold nanoparticles(AuNPs), into a protein-repelling matrix material. It was found thaton homogeneous surfaces, GOx activity is generally lower than thatin its native state and strongly affected by surface chemistry. Lossof activity is related to an increasing amount of β-sheets inthe GOx secondary structure and a corresponding reduction of α-helicalelements. In contrast, on AuNP surfaces, the effect of surface chemistryis negligible, and the amount of adsorbed protein only depends onparticle size. The low activity of GOx on all nanostructures studiedis again accompanied by an increase of β-sheet and a reductionof α-helical secondary structure. The major cause for proteinunfolding on AuNPs thus seems to be the curvature of the surface.In addition, the data suggest that unfavorable orientation of theadsorbed enzyme also contributes to the loss of activity. [ABSTRACT FROM AUTHOR]

Sarcoplasmic reticulum (SR) membranes from rabbit muscle were deposited on silicon substrates and characterized by the combination of spectral ellipsometry (SE), high energy specular X-ray reflectivity (XRR), specular neutron reflectivity (NR), and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Following the optimization of the preparative conditions by SE, the detailed structures in the direction perpendicular to the membrane were probed by XRR. ATR-FTIR data showed strong signals from amide I and amide II bands of the native SR membranes containing a large amount of Ca 2+ -ATPase, which could not be achieved by the reconstitution in artificial lipid membranes. The treatment with protease led to a significant decrease in the amide peaks, and the XRR data confirmed the modulation of the membrane structures. The obtained data show the potential of the in situ combination of reflectivity and vibrational spectroscopy of native supported membranes in order to unravel both structure and dynamics of complex biological membranes.

Osteoarthritis is caused by degeneration of the cartilage, which covers the bone ends of the joints and is decorated with an oligolamellar phospholipid (PL) bilayer. The gap between the bone ends is filled with synovial fluid mainly containing hyaluronic acid (HA). HA and PLs are supposed to reduce friction and protect the cartilage from wear in joint movement. However, a detailed understanding of the molecular mechanisms of joint lubrication is still missing. Previously, we found that aqueous solutions of HA and poly(allylamine hydrochloride) (PAH), the latter serving as a polymeric analogue to HA, adsorb onto the headgroups of surface-bound 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) oligobilayers and significantly enhance their stability with respect to shear forces, typically occurring in joint movement. We now investigated the precise location of PAH chains across the lipid films in neutron reflectivity measurements, as bridging of the oligobilayers by polyelectrolytes (PEs) might be the cause for their improved mechanical stability. In a first set of experiments, we used hydrogenated PAH and chain-deuterated DMPC (DMPC- d 54 ) to improve the contrast between the lipids and potentially intruding PAH. However, due to difficulties in distinguishing between incorporation of water and PAH, penetration into the lipid chain region could hardly be proven quantitatively. Therefore, we designed a more elaborate experiment based on mixed films of DMPC- d 54 and hydrogenated DMPC, which is insensitive to water penetration into the films. Beside facilitating a detailed structural characterization of the oligolamellar system, this elaborate approach showed that PAH adsorbs to the DMPC heads and penetrates the lipid tail strata. No PAH was found in the lipid head strata, which excludes bridging of several lipid bilayers by the PE chains. The data are consistent with the assumption that PAH bridges are formed between the headgroups of two adjacent bilayers and contribute to the enhanced mechanical stability.

Neutron reflectivity at the solid/liquid interface offers unique opportunities for resolving the structure-function relationships of interfacial layers in soft matter science. It is a non-destructive technique for detailed analysis of layered structures on molecular length scales, providing thickness, density, roughness, and composition of individual layers or components of adsorbed films. However, there are also some well-known limitations of this method, such as the lack of chemical information, the difficulties in determining large layer thicknesses, and the limited time resolution. We have addressed these shortcomings by designing and implementing a portable sample environment for in situ characterization at neutron reflectometry beamlines, integrating infrared spectroscopy under attenuated total reflection for determination of molecular entities and their conformation, and spectroscopic ellipsometry for rapid and independent measurement of layer thicknesses and refractive indices. The utility of this combined setup is demonstrated by two projects investigating (a) pH-dependent swelling of polyelectrolyte layers and (b) the impact of nanoparticles on lipid membranes to identify potential mechanisms of nanotoxicity.