Helge Pfeiffer

Fiber optic SPR biosensing of DNA hybridization and DNA–protein interactions

In this paper we present a fiber optic surface plasmon resonance (SPR) sensor as a reusable, cost-effective and label free biosensor for measuring DNA hybridization and DNA-protein interactions. This is the first paper that combines the concept of a fiber-based SPR system with DNA aptamer bioreceptors. The fibers were sputtered with a 50nm gold layer which was then covered with a protein repulsive self-assembled monolayer of mixed polyethylene glycol (PEG). Streptavidin was attached to the PEGs carboxyl groups to serve as a versatile binding element for biotinylated ssDNA. The ssDNA coated SPR fibers were first evaluated as a nucleic acid biosensor through a DNA-DNA hybridization assay for a random 37-mer ssDNA. This single stranded DNA showed a 15 nucleotides overlap with the receptor ssDNA on the SPR fiber. A linear calibration curve was observed in 0.5-5 microM range. A negative control test did not reveal any significant non-specific binding, and the biosensor was easily regenerated. In a second assay the fiber optic SPR biosensor was functionalized with ssDNA aptamers against human immunoglobulin E. Limits of detection (2nM) and quantification (6nM) in the low nanomolar range were observed. The presented biosensor was not only useful for DNA and protein quantification purposes, but also to reveal the binding kinetics occurring at the sensor surface. The dissociation constant between aptamer and hIgE was equal to 30.9+/-2.9nM. The observed kinetics fully comply with most data from the literature and were also confirmed by own control measurements.

Optothermal depth profiling by neural network infrared radiometry signal recognition

The feasibility of a neural network radiometric photothermal depth profiling method is verified using well-defined artificial samples with varying optical properties across the layers. The signal calculation model is shown to be accurate and the neural network approach to solve the inverse problem is shown to be feasible. Both from simulated and experimental radiometric signals, accurate reconstructions are obtained for heat source and optical-absorption coefficient profiles.

Phase Transitions of Binary Lipid Mixtures: A Combined Study by Adiabatic Scanning Calorimetry and Quartz Crystal Microbalance with Dissipation Monitoring

The phase transitions of binary lipid mixtures are studied by a combination of Peltier-element-based adiabatic scanning calorimetry (pASC) and quartz crystal microbalance with dissipation monitoring (QCM-D). pASC, a novel type of calorimeter, provides valuable and unambiguous information on the heat capacity and the enthalpy, whereas QCM-D is proposed as a genuine way of determining phase diagrams by analysing the temperature dependence of the viscosity. Two binary mixtures of phospholipids with the same polar head and differing in the alkyl chain length, DMPC

Apparent sound velocity of lysozyme in aqueous solutions

The reciprocal sound velocity of an ideal solution is, according to the model of Natta and Baccaredda, composed of the reciprocal sound velocities of its components weighed by their volume fractions. This enables us to extract the apparent sound velocity of solutes, and we applied this procedure to a lysozyme solution. With ultrasonic velocimetry, we have obtained an apparent sound velocity of 1958 m/s which is in good agreement with values determined by the ultrasonic pulse-echo method for hydrated lysozyme crystals.

Peculiarity of aqueous solutions of 2,2,2-trifluoroethanol.

Aqueous solutions of 2,2,2-trifluoroethanol appear to show a structural transition at alcohol mole fraction equal to x(TFE) = 0.05, which can be concluded from a discontinuity of the speed of sound. At the same concentration, a discontinuity was observed in the parameters of the long-living component of the positron annihilation spectrum. Moreover, the partial molar volumes of components show transition-like behavior in the range of low solute contents, which is significantly different from nonsubstituted ethanol. The peculiarities of the low concentration system correlate with minor infrared spectra changes assigned to a mode composed of the CH(2) bending and CF(3) stretching internal vibrations being sensitive to polarity of the hydration shell surrounding the solute. The majority of the spectral changes arise from a gradual shift of the equilibrium between trans ↔ gauche isomers when the composition of the solution is changing. A possible explanation for the peculiar behavior of the system is a thermodynamic equilibrium between hydrated monomers and dimers at that respective mole number.

Hydration pressure of a homologous series of nonionic alkyl hydroxyoligo(ethylene oxide) surfactants

The hydration pressure of a homologous series of nonionic surfactants of the type CH3(CH2)n−1(OCH2CH2)mOH (CnEm) was determined by using sorption gravimetry. The hydration pressure shows a non-exponential decay on hydration and the curves are best fitted with a rational function. The non-exponential character is explained by contributions arising from the bending energy of non-lamellar liquid crystalline phases. The parameters obtained show that hydration of pure nonionic surfactants is correlated with the number of the oxyethylene groups in the headgroup. However, they are almost independent of the length of the alkyl chain. Exceptions are surfactants in the solid crystalline state that almost prevent hydration. Furthermore, the respective mesophase structures do not dominate the hydration behaviour. The Gibbs free energy (free enthalpy) of hydration is −1.1 kJ mol−1 per oxyethylene group.

A novel technique for acoustic emission monitoring in civil structures with global fiber optic sensors

The application of acoustic emission (AE)-based damage detection is gaining interest in the field of civil structural health monitoring. Damage progress can be detected and located in real time and the recorded AEs hold information on the fracture process which produced them. One of the drawbacks for on-site application in large-scale concrete and masonry structures is the relatively high attenuation of the ultrasonic signal, which limits the detection range of the AE sensors. Consequently, a large number of point sensors are required to cover a certain area. To tackle this issue, a global damage detection system, based on AE detection with a polarization-modulated, single mode fiber optic sensor (FOS), has been developed. The sensing principle, data acquisition and analysis in time and frequency domain are presented. During experimental investigations, this AE-FOS is applied for the first time as a global sensor for the detection of crack-induced AEs in a full-scale concrete beam. Damage progress is monitored during a cyclic four-point bending test and the AE activity, detected with the FOS, is related to the subsequent stages of damage progress in the concrete element. The results obtained with the AE-FOS are successfully linked to the mechanical behavior of the concrete beam and a qualitative correspondence is found with AE data obtained by a commercial system.

The influence of correlated protein–water volume fluctuations on the apparent compressibility of proteins determined by ultrasonic velocimetry

The elasticity of proteins, expressed by the compressibility, is potentially one of the most important properties of proteins because of the close relationship with its functionality. The compressibility of solutions can be determined by measurements of sound velocity and density. These quantities are related by the Newton-Laplace equation. In order to interpret the apparent compressibility of solutes in highly dilute solutions, it is required to consider the relation between compressibility and sound velocity of the solution using an appropriate reference system. The classical approach usually gives too small values for the apparent compressibility when compared with other methods. We show that the difference can partially be explained if the correlated volume fluctuations of the solvent are taken into consideration. A special attention is given to the compressibility of proteins. Finally, the present paper is not intended to replace established approaches, but it wants to create awareness that the classical mixing rules refer to ideal gasses assuming uncorrelated volume fluctuations and that a considerable part of the hydration effects could be explained by correlated volume fluctuations.

Pressure-induced amorphization in biopolymers

Pressure-induced unfolding of proteins in solution shows analogies to the pressure-induced amorphization observed in some inorganic and polymer systems. More specifically, pressure gives rise to conformations that show a strong tendency to form supramolecular aggregates that have some relevance to molecular diseases. Hydrostatic pressure can tune the conformation of the intermediates along the unfolding/aggregation pathway. Pressure can also be used to probe the stability of the aggregate, and thus the interactions that are responsible for it. In particular, we demonstrate that pressure might be an interesting tool to study the fibril formation. Fourier transform infrared spectroscopy reveals the presence of fibril secondary structures other than random coil and intermolecular β-sheet.

A spectroscopic study of the chromatic properties of GafChromic™EBT3 films

PURPOSE This work provides an interpretation of the chromatic properties of GafChromicEBT3 films based on the chemical nature of the polydiacetylene (PDA) molecules formed upon interaction with ionizing radiation. The EBT3 films become optically less transparent with increasing radiation dose as a result of the radiation-induced polymerization of diacetylene monomers. In contrast to empirical quantification of the chromatic properties, less attention has been given to the underlying molecular mechanism that induces the strong decrease in transparency. METHODS Unlaminated GafChromicEBT3 films were irradiated with a 6 MV photon beam to dose levels up to 20 Gy. The optical absorption properties of the films were investigated using visible (vis) spectroscopy. The presence of PDA molecules in the active layer of the EBT3 films was investigated using Raman spectroscopy, which probes the vibrational modes of the molecules in the layer. The vibrational modes assigned to PDAs were used in a theoretical vis-absorption model to fit our experimental vis-absorption spectra. From the fit parameters, one can assess the relative contribution of different PDA conformations and the length distribution of PDAs in the film. RESULTS Vis-spectroscopy shows that the optical density increases with dose in the full region of the visible spectrum. The Raman spectrum is dominated by two vibrational modes, most notably by the ν(C≡C) and the ν(C=C) stretching modes of the PDA backbone. By fitting the vis-absorption model to experimental spectra, it is found that the active layer contains two distinct PDA conformations with different absorption properties and reaction kinetics. Furthermore, the mean PDA conjugation length is found to be 2-3 orders of magnitude smaller than the crystals PDAs are embedded in. CONCLUSIONS Vis- and Raman spectroscopy provided more insight into the molecular nature of the radiochromic properties of EBT3 films through the identification of the excited states of PDA and the presence of two PDA conformations. The improved knowledge on the molecular composition of EBT3s active layer provides a framework for future fundamental modeling of the dose-response.