Dieter Baurecht

Quantitative modulated excitation Fourier transform infrared spectroscopy

The detection of weak absorption changes induced by an external excitation is often hindered by intense background absorptions as well as by noise. Modulation spectroscopy is an adequate tool to be applied in such a case, provided the system may be periodically stimulated, leading to a periodic reversible or pseudoreversible response. In modulated excitation (ME) Fourier transform infrared spectroscopy the phase sensitive detection (PSD) used for the demodulation of the periodic system response is generally performed during data acquisition, i.e., applied to the intensity of the interferogram. This leads to a number of problems in quantitative analysis and the requirement of optional equipment. In this article, a method is presented to perform an off-line vector PSD of conventional time-resolved spectra after data acquisition. A detailed mathematical analysis of PSD applied to the spectral intensity, the interferogram intensity, and to time-resolved spectra is presented. It is shown, that vector PSD applied to a set of time-resolved spectra is straightforward and avoids any additional mathematical corrections. Furthermore, it will be shown how ME spectroscopy can be used for experimental separation of overlapping bands and a detailed description for the determination of absolute modulation amplitudes and phase lags is given.The detection of weak absorption changes induced by an external excitation is often hindered by intense background absorptions as well as by noise. Modulation spectroscopy is an adequate tool to be applied in such a case, provided the system may be periodically stimulated, leading to a periodic reversible or pseudoreversible response. In modulated excitation (ME) Fourier transform infrared spectroscopy the phase sensitive detection (PSD) used for the demodulation of the periodic system response is generally performed during data acquisition, i.e., applied to the intensity of the interferogram. This leads to a number of problems in quantitative analysis and the requirement of optional equipment. In this article, a method is presented to perform an off-line vector PSD of conventional time-resolved spectra after data acquisition. A detailed mathematical analysis of PSD applied to the spectral intensity, the interferogram intensity, and to time-resolved spectra is presented. It is shown, that vector PSD appli...

A new method of phase sensitive detection in modulation spectroscopy applied to temperature induced folding and unfolding of RNase A

Abstract Modulation spectroscopy can be an adequate tool to separate a weak system response from a huge background absorption, provided the process under consideration enables a periodic external stimulation. Phase sensitive detection (PSD) is then used to demodulate the periodic system response. We introduce a new method of PSD that can be used in modulated excitation (ME)-FTIR spectroscopy without the need of a lock-in amplifier or spectrometer build-in hardware. An advantage of this method is that only standard procedures included in all FTIR instruments, such as the measurement of time-resolved spectra, are required. The principal mathematical formalism of the used PSD is described. As an example we show phase-resolved spectra of the amid I ′ region obtained from temperature modulated FTIR spectroscopic experiments of RNase A. The advantage of the ME compared to a relaxation process is shown by the power of separation of overlapping absorption bands.

Complete Exchange of the Hydrophobic Dispersant Shell on Monodisperse Superparamagnetic Iron Oxide Nanoparticles

High-temperature synthesized monodisperse superparamagnetic iron oxide nanoparticles are obtained with a strongly bound ligand shell of oleic acid and its decomposition products. Most applications require a stable presentation of a defined surface chemistry; therefore, the native shell has to be completely exchanged for dispersants with irreversible affinity to the nanoparticle surface. We evaluate by attenuated total reflectance−Fourier transform infrared spectroscopy (ATR−FTIR) and thermogravimetric analysis/differential scanning calorimetry (TGA/DSC) the limitations of commonly used approaches. A mechanism and multiple exchange scheme that attains the goal of complete and irreversible ligand replacement on monodisperse nanoparticles of various sizes is presented. The obtained hydrophobic nanoparticles are ideally suited for magnetically controlled drug delivery and membrane applications and for the investigation of fundamental interfacial properties of ultrasmall core–shell architectures.

Vibratory communication in spiders

Summary1.Both amplitude and frequency contents of male courtship vibrations of Cupiennius salei change with signal propagation through a bromeliad (Fig. 3). Temporal pattern and carrier frequency of the opisthosomal signal (Fig. 1a) remain largely unchanged, however, and therefore satisfy the requirements to carry species specific information. Pedipalpal signals cover a broad range of frequencies (Fig. 3a, c); both their dispersive transmission and the frequency-dependent attenuation by the plants may provide the female with information about her distance from the male.2.Parallel processing of different signal components already begins in the females metatarsal lyriform vibration receptor. Opisthosomal signals: They mainly elicit responses from long distal slits; signal amplitude is of only minor influence (Fig. 7). Carrier frequency is represented by interspike frequencies of individual slits (Fig. 8b, d). Pedipalpal signals: They elicit responses from all slits (Figs. 6, 7a). Responses of individual slits differ and can be assigned to specific frequency components contained in a pedipalpal signal (Figs. 6, 8a, 9b).3.The repetition of opisthosomal signals within the male courtship vibration improves the signal to noise ratio in the females receptor response (Fig. 10).4.For unknown reasons the intensity of the receptor response follows changes in acceleration amplitude in the case of heterospecific but not in the case of conspecific courtship vibrations (Fig. 11).

Validation of the combined ATR-FTIR/tape stripping technique for monitoring the distribution of surfactants in the stratum corneum

The physical presence of surfactants in the skin is linked to their skin irritation potential. Combined ATR-FTIR spectroscopy and tape stripping experiments in vitro on porcine ear skin were used to investigate the spatial distribution of sodium lauryl ether sulfate (SLES) in the stratum corneum and to assess its effects on conformational order of stratum corneum intercellular lipids, secondary structure of keratin and skin hydration. It was possible to monitor the spatial distribution of SLES in the stratum corneum for the first time by subtracting spectra of untreated from treated skin samples and without the need of a perdeuterated form. This method of analysis was evaluated by addressing potential error sources such as differences in removed amounts of corneocytes and intra-individual changes in stratum corneum composition as a function of depth. The obtained results indicate a penetration of SLES into deep layers of the stratum corneum. Furthermore, SLES treatment led to significantly decreased skin hydration levels, whereas the secondary structure of keratin remained nearly unaffected. The reliability of this semi-quantitative method of analysis was confirmed by receiving a coefficient of determination of 0.9963 after making a correlation of deep depended absorbances of two different characteristic bands with different absorption coefficients.

Conformational transitions and molecular hysteresis of cytochrome c oxidase: Varying the redox state by electronic wiring

Even though the structures of cytochrome c oxidase (CcO) from different sources have been determined by X-ray crystallography in both the reduced and oxidized redox states, information about redox-induced structure-function relationships is still very limited. In the current work, redox-dependent structural changes are determined for CcO reconstituted in a protein-tethered bilayer lipid membrane by surface-enhanced infrared absorption spectroscopy in the ATR mode. Significantly, the redox changes in the enzyme are attained by direct wiring of CcO to a gold electrode, ensuring that sequential intra-protein electron transfer occurs by a directed pathway that is natural to the system. The characteristics of CcO were observed to be dramatically altered after the reconstituted enzyme was allowed to turn over in the presence of O2. The data suggest that the enzyme is initially in an “inactive” state, but that direct electron transfer in the presence of O2 converts the enzyme to an “activated” form which returns to the inactive conformation when the enzyme remains idle under anaerobic conditions. Potentiometric titrations are performed and reduced-minus-fully oxidized and oxidized-minus-fully reduced absorbance spectra are recorded at decreasing and increasing potentials, respectively, applied to the electrode in a regular succession. The two sets of difference spectra show mirror symmetry, however, they markedly differ from those measured in the presence of redox mediators. Plots of band area of individual bands obtained by Fourier self deconvolution vs. applied potential show a sigmoid dependence as expected for a redox process. However, the sigmoid curves do not coincide but are displaced depending on the direction of the potential change. In other words, these curves show hysteresis, which is an indication of cooperativity and non-equilibrium states for electron transfer and/or conformational changes of the protein. This is discussed in terms of known concepts of molecular hysteresis.

Application of special FTIR ATR techniques for quantitative structural analysis of thin surface layers

FTIR ATR spectroscopy is increasingly used for in situ investigations of processes at or near a surface. Particularly when thin layers (biomembranes, monolayers, thin films) are investigated with respect to surface concentration and molecular structure, very sensitive techniques have to be applied in order to achieve an adequate signal-to-noise ratio. This may lead to long measuring times due to extended data accumulation and averaging. However, this can cause new problems with respect to the stability of relevant experimental parameters, such as the sample itself, the spectrometer, and the atmosphere in the spectrometer. In this article we report on two techniques which were developed or improved in our laboratory and successfully applied over past years. Both methods, the so-called single-beam sample reference (SBSR) spectroscopy and the modulation or modulated excitation (ME) spectroscopy, are well suited to compensate instabilities that occur in the course of an experimental series. The SBSR technique converts a single-beam FTIR spectrometer into a pseudo double-beam instrument. By this technique there is always a reference with the same age as the sample available. Moreover, by alternating sample and reference measurements within short time periods, varying environmental conditions such as water vapor concentration in the spectrometer are easily compensated. Moreover SBSR technique enables data evaluation in the conventional single-beam mode (SB) in both the sample (S) and reference (R) channel. This kind of evaluation is important to gain information on the history of S and R. As examples for SBSR and SB applications we report on studies of the interaction of an endotoxin with an immobilized lipid bilayer membrane, as well as on the interaction of TNFa with a TNFa antibody. ME spectroscopy can be applied to systems that show a (pseudo-) reversible response to a periodic excitation. The response of the system measured with time-resolved FTIR spectroscopy is then processed by phase-sensitive detection (PSD). ME spectroscopy is able to determine kinetic constants of a system, allows a hardware separation of overlapping absorption bands, and eliminates all disturbing signal components which do not have the same frequency as the excitation itself. This improves the signal-to-noise ratio dramatically and leads in principal to a stable baseline. The binding of sodium cholate to an adsorbed protein layer of human serum albumin (HSA) is shown as an example that the required sensitivity to study specific molecular interaction is in the μAU range and can be reached by FTIR ME spectroscopy. In a second example, the measurement of structural changes of PLL induced by temperature modulation shows the feasibility of band separation and indicates the possible determination of kinetic properties of a system.

S-layer templated bioinspired synthesis of silica

The current understanding of the molecular mechanisms involved in the bioinspired formation of silica structures laid foundation for investigating the potential of the S-layer protein SbpA from Lysinibacillus sphaericus CCM 2177 as catalyst, template and scaffold for the generation of novel silica architectures. SbpA reassembles into monomolecular lattices with square (p4) lattice symmetry and a lattice constant of 13.1 nm. Silica layers on the S-layer lattice were formed using tetramethoxysilane (TMOS) and visualized by transmission electron microscopy. In situ quartz crystal microbalance with dissipation monitoring (QCM-D) measurements showed the adsorption of silica in dependence on the presence of phosphate in the silicate solution and on the preceding chemical modification of the S-layer. An increased amount of precipitated silica could be observed when K2HPO4/KH2PO4 was present in the solution (pH 7.2). Further on, independent of the presence of phosphate the silica deposition was higher on S-layer lattices upon activation of their carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) compared to native S-layers or EDC treated S-layers when the activated carboxyl groups were blocked with ethylene diamine (EDA). Fourier transform infrared attenuated total reflectance (FTIR-ATR) spectroscopy revealed the formation of an amorphous silica gel (SiO2)x.yH2O on the S-layer. The silica surface concentrations on the S-layer was 4 x 10(-9) to 2 x 10(-8) mol cm(-2) depending on the modification of the protein layer and corresponded to 4-21 monolayers of SiO2.

Electric Field-Induced Changes in Lipids Investigated by Modulated Excitation FTIR Spectroscopy

The effect of electric fields on dry oriented multibilayers of dimyristoylphosphatidylcholine (DMPC) was investigated by transmission Fourier transform infrared electric field modulated excitation (E-ME) spectroscopy. A periodic rectangular electric potential (0-150 V, 1.25 Hz, 28.4 degrees C +/- 0.2 degrees C) was applied across the sample. To discriminate electric field-induced effects from possible temperature-induced effects resulting from a current flow (<1 pA) across the sample, corresponding temperature-modulated excitation (T-ME) measurements within the temperature uncertainty limits of +/-0.2 degrees C at 28.4 degrees C were performed. T-ME induced reversible gauche defects in the hydrocarbon chains, whereas E-ME resulted in reversible compression of dry DMPC bilayers. Periodic variation of the tilt angle of the hydrocarbon chains is suggested. The degree of absorbance modulation in the CH-stretching region was found to be in the order of 1:700, corresponding to a variation of the bilayer thickness of Deltaz = 0.0054 nm. Using a series connection of capacitors as equivalent circuit of the cell resulted in E = (1.2 +/- 0.7) x 10(7) V/m for the electric field in DMPC. Youngs elasticity modulus of DMPC could be calculated to be E( perpendicular ) = 2.2 x 10(6) Pa +/- 1.8 x 10(6) Pa, which is in good agreement with published data obtained by electric field-dependent capacitance measurements.

Determination of surface concentrations of individual molecule-layers used in nanoscale biosensors by in situ ATR-FTIR spectroscopy

For the development of nanowire sensors for chemical and medical detection purposes, the optimal functionalization of the surface is a mandatory component. Quantitative ATR-FTIR spectroscopy was used in situ to investigate the step-by-step layer formation of typical functionalization protocols and to determine the respective molecule surface concentrations. BSA, anti-TNF-α and anti-PSA antibodies were bound via 3-(trimethoxy)butylsilyl aldehyde linkers to silicon-oxide surfaces in order to investigate surface functionalization of nanowires. Maximum determined surface concentrations were 7.17 × 10(-13) mol cm(-2) for BSA, 1.7 × 10(-13) mol cm(-2) for anti-TNF-α antibody, 6.1 × 10(-13) mol cm(-2) for anti-PSA antibody, 3.88 × 10(-13) mol cm(-2) for TNF-α and 7.0 × 10(-13) mol cm(-2) for PSA. Furthermore we performed antibody-antigen binding experiments and determined the specific binding ratios. The maximum possible ratio of 2 was obtained at bulk concentrations of the antigen in the μg ml(-1) range for TNF-α and PSA.