BioRef II-Neutron reflectometry with relaxed resolution for fast, kinetic measurements at HZB.

We present an upgrade to the time-of-flight neutron reflectometer BioRef at the research reactor BER II of the Helmholtz-Zentrum Berlin für Materialien und Energie (HZB). Through the integration of an additional chopper into the existing setup, the available wavelength resolution is significantly extended. Now two distinct operation modes can be used: a high resolution mode with Δλ/λ ranging from 1% to 5%, which allows for the investigation of thick films up to 4000 Å, and a high flux mode with Δλ/λ = 7%-11%. In the high flux mode, reflectivity curves from 0.007 Å-1 to 0.2 Å-1 with three angular settings can be recorded in 7 min. For a single angular setting and its respective window in Q-space, a time resolution of even less than 4 min is reached. The different configurations are documented by respective measurements (a) on a Ni-Ti multilayer and (b) the swelling kinetics of a solid-supported phospholipid coating upon incubation in a polyelectrolyte solution.

Localization microscopy (SPDM) facilitates high precision control of lithographically produced nanostructures.

Nanoscale resolution in material sciences is usually restricted to scanning electron beam microscopes. Here we present a procedure that allows single molecule resolution of the sample surface with visible light. Highlighting the performance we used electron beam lithography to generate highly regular nanostructures consisting of interconnected cubes. The samples were labeled with Alexa 647 dyes. The spatial organization of the dyes on nanostructured surfaces was localized with single molecule resolution using localization microscopy. This succeeded also in an absolute spatial calibration of the localization method applied (spectral precision distance microscopy/SPDM). The findings will contribute to the field of product control for industrial applications and long-term fluorescence imaging.

Conformation and activity of glucose oxidase on homogeneously coated and nanostructured surfaces.

Protein unfolding and loss of protein function upon surface contact is a major problem in biotechnology and biomedicine. Using glucose oxidase (GOx) as a model protein, we investigated the impact of surface chemistry, topography, and confinement on enzyme activity, conformation, and affinity. A particular focus lay on the question whether the conformation of surface-bound proteins can be stabilized by embedding nanoscale adsorption sites, here in the form of monodisperse gold nanoparticles (AuNPs), into a protein-repelling matrix material. It was found that on homogeneous surfaces, GOx activity is generally lower than that in its native state and strongly affected by surface chemistry. Loss of activity is related to an increasing amount of ß-sheets in the GOx secondary structure and a corresponding reduction of α-helical elements. In contrast, on AuNP surfaces, the effect of surface chemistry is negligible, and the amount of adsorbed protein only depends on particle size. The low activity of GOx on all nanostructures studied is again accompanied by an increase of ß-sheet and a reduction of α-helical secondary structure. The major cause for protein unfolding on AuNPs thus seems to be the curvature of the surface. In addition, the data suggest that unfavorable orientation of the adsorbed enzyme also contributes to the loss of activity.

Impact of a model synovial fluid on supported lipid membranes.

The interaction of a model synovial fluid, here a solution of 3mg/mL hyaluronic acid (HA) in heavy water (D(2)O), with an oligolamellar stack of lipid (DMPC) membranes on silicon support has been studied by neutron reflectometry and infrared spectroscopy on the molecular scale at non-physiological and physiological conditions. The system under investigation represents a simple model for lipid-coated mammalian joints and other artificial implant surfaces. When exposed to pure D(2)O at 21°C, i.e. below the main phase transition of the system, the lipid membranes show a lamellar spacing of 65Å. Heating to 26°C results in detachment of all lipid bilayers except for the innermost lipid lamella directly adsorbed to the surface of the silicon support. On the contrary, when incubated in the solution of HA in D(2)O the oligolamellar lipid system starts swelling. In addition, heating to 39°C does not result in loss of the lipid membranes into the liquid phase. The interfacial lipid coating adopts a new stable lamellar state with an increase in d-spacing by 380% to 247Å measured after 43 days of incubation with the model synovial fluid. Potential consequences for joint lubrication and protective wear functionality are considered.

BioRef: a versatile time-of-flight reflectometer for soft matter applications at Helmholtz-Zentrum Berlin.

BioRef is a versatile novel time-of-flight reflectometer featuring a sample environment for in situ infrared spectroscopy at the reactor neutron source BER II of the Helmholtz Zentrum Berlin für Materialien und Energie (HZB). After two years of design and construction phase the instrument has recently undergone commissioning and is now available for specular and off-specular neutron reflectivity measurements. BioRef is especially dedicated to the investigation of soft matter systems and studies at the solid-liquid interface. Due to flexible resolution modes and variable addressable wavelength bands that allow for focusing onto a selected scattering vector range, BioRef enables a broad range of surface and interface investigations and even kinetic studies with subsecond time resolution. The instrumental settings can be tailored to the specific requirements of a wide range of applications. The performance is demonstrated by several reference measurements, and the unique option of in situ on-board infrared spectroscopy is illustrated by the example of a phase transition study in a lipid multilayer film.

Protein density profile at the interface of water with oligo(ethylene glycol) self-assembled monolayers.

We determined the density profile of a high-molecular-weight globular protein (bovine serum albumin, BSA) solution at the methoxy tri(ethylene glycol)-terminated undecanethiol SAM/protein solution interface by neutron reflectivity measurements. Information about the interactions between oligo(ethylene glycol) (OEG)-terminated self-assembled monolayers (SAMs) and proteins is derived from the analysis of the structure of the solid-liquid interface. The fitting results reveal oscillations of the protein density around the bulk value with decaying amplitude on a length scale of 4 to 5 nm. The amplitude, phase, period, and decay length are found to vary only slightly with temperature and the ionic strength of the protein solution. Adsorption is reversible within the limits of detection, which suggests that the hydrated ethylene glycol surface inhibits the protein from unfolding and irreversible bonding. The insensitivity of BSA adsorption toward the ionic strength of the solution contrasts with observations in surface force experiments with a fibrinogen-coated AFM tip, where electrostatic repulsion dominates theprotein/OEG SAM interaction. As reported previously, irreversible BSA adsorption takes place below 283 K, which we interpret as indicative of the presence of dynamic effects in the protein resistance of short-chain OEG-terminated surfaces.

Label-free biosensors based on optically responsive nanocomposite layers: sensitivity and dynamic range.

Core-shell nanoparticle layers have proven to be a promising tool for the label-free detection of binding events. Upon reflection of white light, they exhibit pronounced extinction peaks in the UV/vis and NIR regime of the electromagnetic spectrum, which shift to higher wavelengths when molecules are adsorbed. Beside drastic simplification of the instrumentation and related reduction in cost, a significantly stronger response toward alkanethiol adsorption has been observed in previous experiments than in conventional surface plasmon resonance (SPR). However, as the amount of molecules deposited onto the nanoparticle films was unknown, no quantitative relationship could be established between the measured wavelength shifts and the surface mass density of the adsorbate. In order to facilitate quantitative molecule detection, self-assembled monolayers (SAMs) of simple and ethylene glycol (EG) terminated alkanethiols with various chain lengths were prepared on the nanoparticle-coated substrates. The measured red-shift of the extinction spectrum upon molecule adsorption was related to the amount of adsorbate as determined by X-ray photoelectron spectroscopy (XPS). For the whole range of film thicknesses studied, a linear relationship is found yielding a sensitivity factor of 0.027 nm/(ng/cm (2)). As proven by enzyme-linked immunosorbent assay (ELISA), such determined sensitivity factor can also be used to correctly predict the amount of surface-bound protein in immunoreactions from the measured wavelength shifts. It is concluded that the decay length of the evanescent electric field associated with the nanoparticle sensors is more than 100 nm and, thus, significantly larger than that observed for localized surface plasmons excited in small isolated metal clusters.

New sources and instrumentation for neutrons in biology.

Neutron radiation offers significant advantages for the study of biological molecular structure and dynamics. A broad and significant effort towards instrumental and methodological development to facilitate biology experiments at neutron sources worldwide is reviewed.

Characteristics of acoustic plate modes on rotated Y-cuts of quartz utilized for biosensing applications.

Acoustic plate modes (APM) on various quartz substrates have been investigated in order to determine their usefulness for liquid-sensing applications. The modes have been characterized in terms of their mass sensitivity, mode separation, temperature sensitivity, and reproducibility of the experimental results. Promising characteristics are found for rotated Y-cuts of quartz with the direction of acoustic mode propagation being perpendicular to the X-axis of the quartz crystal. Experiments on the detection of immunochemical reactions are performed using different quartz APM sensors, and the results are compared to similar experiments utilizing APM devices on ZX-LiNbO3.

Operation of acoustic plate mode immunosensors in complex biological media.

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 non-specific 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.

Acoustic wave-based sensors using mode conversion in periodic gratings.

Using periodic gratings etched into the surface of a piezoelectric plate, surface acoustic waves (SAW) can be converted into bulk waves and vice versa with high efficiency. If parallel grating structures are fabricated on opposite surfaces of a piezoelectric plate, a SAW also can be directed from one surface to the other. Using such structures, acoustic wave-based sensors can be designed that utilize SAW for the detection of chemical analytes on an electrode-free surface, i.e., the back surface. As a result, spurious sensor response and electrode aging that may occur when a chemical analyte comes in contact with the transducers are minimized. The design principles of these grating-based SAW sensors are explained, and the mass sensitivity is investigated using chemical vapor deposited thin polymer films, a type of material used in many practical chemical sensor applications. Experimental results are presented for the detection of nitrogen dioxide (NO(2 )) in sub-ppm concentrations.

A concentration dependent study of acoustic plate mode immunosensor response using antigen/antibody systems with different binding ability.

Acoustic plate mode sensors have been used to monitor immunochemical reactions as a function of antigen concentration. In the studies, antibodies were covalently linked to the gold-coated sensing surface via mercaptoethanol, aminosilane, and glutaraldehyde. Two antigen/antibody model systems that differ in their ability to mutually bind one another have been used. For sensor operation at about 150 MHz, a detection limit of approximately 0.5 microg/ml was obtained in both cases. No significant difference between the two systems was found for the value of the binding constants. They amount to about 1.10(8) 1/mole and fall well into the range of binding constants reported for homogeneous immunoassays. A comparison of the sensor response obtained for the two model systems shows that about 70% of the immobilized antibodies are active.

On-line detection of nonspecific protein adsorption at artificial surfaces.

A detailed understanding of the interaction of proteins with artificial surfaces is essential for many applications in medicine and biochemistry. The affinity of surfaces toward proteins may, for instance, remove pharmacological proteins from media or control the adherence of pathogenic bacteria to protheses. Only a few analytical techniques now exist that can be used to study the binding process in real time, using unlabeled proteins. By investigating the adsorption kinetics of fibrinogen at differently terminated self-assembled monolayers (SAMs) of alkanethiols on thin gold films, it is demonstrated that acoustic plate-mode sensors are a promising analytical tool for studying the adsorption of proteins. In agreement with previous studies for fibrinogen, it is shown in situ that hexa(ethylene glycol)-terminated SAMs (HS(CH2)11 (OCH2CH2)6OH) exhibit very low protein adsorption and that methyl-terminated SAMs (HS(CH2)11CH3) tend to absorb large amounts of protein nonspecifically. The observed adsorption kinetics deviate from classical Langmuir behavior; these kinetics are compatible with a mechanism that involves an unfolding of fibrinogen after adsorption. Film quality is controlled by IR, XPS, and contact angle measurements.

Multifrequency evaluation of different immunosorbents on acoustic plate mode sensors.

Previous studies of acoustic plate modes on ZX-LiNbO3 have indicated that practical mass-sensitive immunosensors can be implemented by using devices with higher frequencies of operation and/or by improving techniques for the immobilization of antibodies. However, it is also known from these studies that the viscoelastic properties of aminosilane films, used for the covalent immobilization of antibodies on the crystal surface, cannot be ignored in the sensor response. In the present work, in an attempt to study the effect of viscoelasticity of the binding film, three different films with different viscoelasticity and binding capacities, an aminosilane, a dextran, and a poly-(etherurethane)-based immunosorbent (XP-5), were prepared on the sensor surface for the immobilization of antibodies. Immunochemical reactions were monitored by the acoustic plate mode sensor at three different frequencies, thus allowing the direct observation of the frequency dependence of mass sensitivity with different films. Depending on the type of immunosorbent, the sensitivity at the third harmonic was enhanced by a factor of 2-5 with respect to the fundamental response. A third acoustic mode at a closely spaced frequency to the third harmonic yielded lower sensitivity values, which indicates that sensitivity depends not only on the frequency of device operation but also on particle displacement amplitude and components of the selected wave. Since antigen binding capacities of the different immunosorbents were determined independently by a modified ELISA test, sensor responses can also be correlated to the immunosorbent structure, and hence the viscoelastic properties. A dual delay line configuration was used which compensates for second-order effects such as temperature variations and nonspecific adsorption.

Identification of metal ion solutions using acoustic plate mode devices and pattern recognition.

The temperature dependence of acoustic plate mode (APM) devices used as probes for dilute electrolytes is described. Specifically, the probe responses that consist of the frequency change and device loss were studied for dilute aqueous solutions of alkali metal ions. It is shown that by integrating the temperature dependence of the APM probe responses with pattern recognition techniques, valuable information about the solutions can be obtained that include identification and quantification. A preliminary investigation of the feasibility of identifying binary mixtures of the alkali metal ion solutions using only the temperature responses showed good results.