Analytical and Bioanalytical Chemistry (v.390, #6)

14th Conference on Solid State Analysis by Gernot Friedbacher (1437-1438).
is Professor of Analytical Chemistry at the Institute of Chemical Technologies and Analytics, Vienna University of Technology. He is author and co-author of over 100 publications. His research interests are focused on surface and interface analysis with scanning probe microscopy and electron probe microanalysis.

Application of high-resolution EFTEM SI in an AEM by Bernhard Schaffer; Werner Grogger; Gerald Kothleitner; Ferdinand Hofer (1439-1445).
In this work we show how energy-filtered imaging can be used to obtain spectrum images of electron energy-loss spectrometric data. Focus is placed on improved energy resolution within these data sets. Using two multilayer samples (GaN/AlN and InP/InAs), we demonstrate the advantages of spectrum-imaging and its extended mapping capabilities. Plasmon-ratio maps are used to quickly create high-contrast material maps with high signal-to-noise ratio, ratio-contrast plots are used to gain optimum settings for the ratio maps, and plasmon-position maps are used to map small shifts of the energy position of bulk plasmon peaks. Figure Scheme of EELS SI and derived plasman-position map
Keywords: Spectrum-imaging; Energy-filtered transmission electron microscopy; Electron energy loss spectrometry; GaN/AlN; InP/InAs

N-K electron energy-loss near-edge structures for TiN/VN layers: an ab initio and experimental study by Petr Lazar; Josef Redinger; Johannes Strobl; Raimund Podloucky; Boriana Rashkova; Gerhard Dehm; Gerald Kothleitner; Sašo Šturm; Kerstin Kutschej; Christian Mitterer; Christina Scheu (1447-1453).
We study N-K-edge electron energy-loss near-edge structures for well-defined TiN/VN bilayers grown on a MgO(100) substrate by both calculations and experiments. The structural relaxations and the electronic structure of TiN/VN multilayers are calculated using the Vienna Ab Initio Simulation Package computer code, which uses density functional theory to describe the electronic interaction. The effects of the core hole created in the excitation process are included in the calculations. For VN, off-stoichiometric effects due to nitrogen vacancies are modelled. The partial density of states (PDOS) for the N-K edge of atoms in the vicinity of the TiN/MgO interface revealed that two new peaks appear between 7 and 9 eV instead of a broad shoulder typical for the bulk. For the VN/TiN interface, the PDOS is modified only slightly, owing to similar bonding on both sides of the interface, and is thus very similar to the respective bulk spectra. An experimental spectrum taken at the VN/TiN interface is, however, well described by an average of the simulated spectra for VN and TiN bulk (interface). Such a finding is characteristic of an intermixed interface.
Keywords: Spectroscopy/theory; Interface/surface analysis; Thin films

AFM, ellipsometry, XPS and TEM on ultra-thin oxide/polymer nanocomposite layers in organic thin film transistors by A. Fian; A. Haase; B. Stadlober; G. Jakopic; N. B. Matsko; W. Grogger; G. Leising (1455-1461).
Here we report on the fabrication and characterization of ultra-thin nanocomposite layers used as gate dielectric in low-voltage and high-performance flexible organic thin film transistors (oTFTs). Reactive sputtered zirconia layers were deposited with low thermal exposure of the substrate and the resulting porous oxide films with high leakage currents were spin-coated with an additional layer of poly-α-methylstyrene (PαMS). After this treatment a strong improvement of the oTFT performance could be observed; leakage currents could be eliminated almost completely. In ellipsometric studies a higher refractive index of the ZrO2/PαMS layers compared to the “as sputtered” zirconia films could be detected without a significant enhancement of the film thickness. Atomic force microscopy (AFM) measurements of the surface topography clearly showed a surface smoothing after the PαMS coating. Further studies with X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM) also indicated that the polymer definitely did not form an extra layer. The polymer chains rather (self-)assemble in the nano-scaled interspaces of the porous oxide film giving an oxide–polymer “nanocomposite” with a high oxide filling grade resulting in high dielectric constants larger than 15. The dielectric strength of more than 1 MV cm−1 is in good accordance with the polymer-filled interspaces.
Keywords: Organic thin film transistor; oTFT; Gate dielectric; Zirconia; Ellipsometry

Smoothing and passivation of special Si(111) substrates: studied by SPV, PL, AFM and SEM measurements by H. Angermann; J. Rappich; I. Sieber; K. Hübener; J. Hauschild (1463-1470).
Surface sensitive techniques, the field-modulated surface photovoltage, photoluminescence measurements, atomic force microscopy and scanning electron microscopy, were employed to yield detailed information on the influence of wet-chemical treatments on the preparation induced microroughness and electronic properties of wet-chemically passivated Si(111) substrates with special surface morphology. Stepped substrates with evenly distributed atomically flat terraces were prepared and passivated by thin oxide layers, which were used as a starting point for the subsequent H-termination after long storage in air. It was shown that their surface morphology and electronic properties do not degrade. Applying this preparation method to solar cell substrates with randomly distributed Si(111) pyramids, we achieved significantly lower densities of surface states and reduced recombination loss at a-Si:H/c-Si interfaces, compared with conventional pretreatments. The surface microroughness, the density of rechargeable states and the resulting recombination loss on a-Si:H/c-Si heterojunctions were found to be mainly influenced by two steps of surface pretreatment: firstly, the wet-chemical smoothing procedure of structured substrates and, secondly, the removal of native and wet-chemical oxides during the final etching in HF- or NH4F- containing solutions. Figure After wet-chemical oxidation in H2SO4/H2O2 and storage in air
Keywords: Surface photovoltage; Photoluminescence; Atomic force microscopy; Electron microscopy; Interface state density; Wet-chemical surface pretreatment

Infrared spectroscopy is often used to monitor the formation of the perovskite phase during the preparation of lead zirconate titanate (PZT) thin films and fibres and also to detect phase transitions. Infrared spectroscopy has rarely been used to investigate bulk samples or thick films of PZT. In this study, the first results of infrared investigations of microwave-sintered PZT and PZT reinforced with powdered copper are presented and compared with results from thermally heated samples. The infrared spectra show the typical Zr/TiO6 metal–oxygen octahedral vibrational modes in the range 750 to 450 cm−1. This band is broadened for the pure PZT samples at higher sintering temperatures. A shift of the peak to lower wave numbers with increasing temperatures can be proven for pure PZT samples, but not for the Cu-reinforced PZT samples.
Keywords: Lead zirconate titanate; Microwave sintering; Infrared spectroscopy; X-ray diffraction

Application of FTIR spectra for evaluating interfacial reactions in metal matrix composites by S. Kúdela Jr.; S. Oswald; S. Kúdela; K. Wetzig (1477-1486).
Manufacturing of Saffil/MgLi metal matrix composites by the melt infiltration process is accompanied by extensive interfacial redox reaction between δ-Al2O3 fibers (Saffil) and lithium. The present paper deals with the Fourier transform infrared spectroscopy examination of Saffil fibers isolated from Mg–8 wt% Li alloy by the bromine/methylacetate agent focusing on the insertion of Li+ ions into δ-Al2O3 and their influence on water adsorption. Insertion of Li+ into δ-Al2O3 is monitored by gradual change of Al–O stretching bands (400–900 cm−1) towards more simple patterns of a spinel-like product assigned as δ(Li) which transforms to LiAl5O8 during subsequent annealing. Rapid increase in the water adsorption with increase in Li content, indicated by the changes in H–O–H bending (about 1,650 cm−1) and O–H stretching (about 3,500 cm−1), is connected with the ionicity of the δ(Li) phase, which attracts polar water molecules.
Keywords: Infrared spectroscopy; Metal matrix composites; Composite interfaces; Transition aluminas; Lithium aluminate; Lithium-doped alumina

In artificial prosthetics for knee, hip, finger or shoulder joints, ultrahigh molecular weight polyethylene (UHMW-PE) is a significant material. Several attempts to reduce the wear rate of UHMW-PE, i.e. the application of suitable coatings, are in progress. A surface modification of polyethylene with wear-resistant hydrogenated diamond-like carbon is favourable, owing to the chemical similarity of polyethylene (–C–H2–) n and C:H or amorphous C:H (a–C:H) coatings with diamond-like properties. In the present study, the microstructure of a–C:H coatings on UHMW-PE substrates was investigated by Raman and Fourier transform infrared (FT-IR) spectroscopy. FT-IR spectroscopy shows very broad absorption lines, which point to the disorder and diversity of different symmetric, asymmetric aromatic, olefin sp 2-hybridized or sp 3-hybridized C–H groups in the amorphous diamond-like carbon coating. Following a long incubation of 12 months in a simulated body liquid, the structural investigations were repeated. Furthermore, fractured cross-sections and the wetting behaviour with polar liquids were examined. After incubation in simulated body liquid, Raman spectroscopy pointed to a reduction of the C–H bonds in the diamond-like carbon coatings. On the basis of these findings, one can conclude that hydrogenated diamond-like carbon is able to interact with salt solutions by substituting the hydrogen with appropriate ions.
Keywords: Diamond-like carbon; Ultrahigh molecular weight polyethylene; Raman spectroscopy; Fourier transform infrared spectroscopy

Influence of surface contaminations on the hydrogen storage behaviour of metal hydride alloys by Mark Schülke; Hubert Paulus; Martin Lammers; Gábor Kiss; Ferenc Réti; Karl-Heinz Müller (1495-1505).
Hydrogen storage in metal hydrides is a promising alternative to common storage methods. The surface of a metal hydride plays an important part in the absorption of hydrogen, since important partial reaction steps take place here. The development of surface contaminations and their influence on hydrogen absorption is examined by means of absorption experiments and surface analysis, using X-ray photoelectron spectroscopy (XPS), thermal desorption mass spectrometry (TDMS) and secondary neutral mass spectrometry (SNMS), in this work. All investigations were carried out on a modern AB2 metal hydride alloy, namely Ti0.96Zr0.04Mn1.43V0.45Fe0.08. Surface analysis (SNMS, XPS) shows that long-term air storage (several months) leads to oxide layers about 15 nm thick, with complete oxidation of all main alloy components. By means of in situ oxygen exposure at room temperature and XPS analysis, it can be shown that an oxygen dose of about 100 Langmuirs produces an oxide layer comparable to that after air storage. Manganese enrichment (segregation) is also clearly observed and is theoretically described here. This oxide layer hinders hydrogen absorption, so an activation procedure is necessary in order to use the full capacity of the metal hydride. This procedure consists of heating (T = 120° C) in vacuum and hydrogen flushing at pressures like p = 18 bar. During the activation process the alloy is pulverized to particles of ∼20 μm through lattice stretches. It is shown that this pulverization of the metal hydride (creating clean surface) during hydrogen flushing is essential for complete activation of the material. Re-activation of powder contaminated by small doses of air (p ≈ 0.1 bar) does not lead to full absorption capacity. In ultrahigh vacuum, hydrogen is only taken up by the alloy after sputtering of the surface (which is done in order to remove oxide layers from it), thus creating adsorption sites for the hydrogen. This is shown by TDMS measurements with and without sputtering and oxygen exposure. Figure Investigated metal hydride before and after activation
Keywords: Hydrogen storage; Metal hydrides; Surface analysis; Oxygen adsorption; Surface contamination; Concentration–pressure isotherms

Nitridation of niobium oxide films by rapid thermal processing by V. A. Matylitskaya; W. Bock; B. O. Kolbesen (1507-1515).
The possibility of forming niobium oxynitride through the nitridation of niobium oxide films in molecular nitrogen by rapid thermal processing (RTP) was investigated. Niobium films 200 and 500 nm thick were deposited via sputtering onto Si(100) wafers covered with a thermally grown SiO2 layer 100 nm thick. These as-deposited films exhibited distinct texture effects. They were processed in two steps using an RTP system. The as-deposited niobium films were first oxidized under an oxygen atmosphere at 450 °C for various periods of time and subsequently nitridated under a nitrogen atmosphere at temperatures ranging from 600 to 1000 °C for 1 min. Investigations of the oxidized films showed that samples where the start of niobium pentoxide formation was detected at the surface and the film bulk still consisted of a substoichiometric NbOx phase exhibited distinctly lower surface roughness and microcrack densities than samples where complete oxidation of the film to Nb2O5 had occurred. The niobium oxide phases formed at the Nb/substrate interface also showed distinct texture. Zones of niobium oxide phases like NbO and NbO2, which did not exist in the initial oxidized films, were formed during the nitridation. This is attributed to a “snow-plough effect” produced by the diffusion of nitrogen into the film, which pushes the oxygen deeper into the film bulk. These oxide phases, in particular the NbO2 zone, act as barriers to the in-diffusion of nitrogen and also inhibit the outdiffusion of oxygen from the SiO2 substrate layer. Nitridation of the partially oxidized niobium films in molecular nitrogen leads to the formation of various niobium oxide and nitride phases, but no indication of niobium oxynitride formation was found. Figure Schematic representation of the phase distribution in 200 nm Nb film on SiO2/Si substrate after two steps annealing using an RTP system. The plot below represents the SIMS depth profiles of the nitridated sample with the phase assignment
Keywords: Niobium; Thin films; Rapid thermal processing (RTP); Niobium oxides; Nitrides; Oxynitrides; Diffusion barrier; X-ray diffraction (XRD); SIMS depth profile

B/Nb and B/Nb2N bilayers and Nb/B/Nb trilayers of about 550 nm total thickness have been deposited on Si(100) wafers with 100 nm thermally grown oxide. Nb and B layers were deposited by magnetron sputtering. Nb2N layers were prepared by nitridation of Nb films via rapid thermal processing (RTP). The samples were annealed subsequently at temperatures between 600 and 1,200 °C in an RTP system under Ar or NH3 gas flow to study interdiffusion and reactivity of niobium, boron and nitrogen. Formation of phases was investigated by X-ray diffraction (XRD); surface morphology and roughness were studied via scanning electron microscopy (SEM) and atomic force microscopy (AFM), respectively. Elemental depth profiles of selected samples were recorded by secondary ion mass spectrometry (SIMS). Annealing of the B/Nb bilayers and Nb/B/Nb trilayers under Ar leads to the formation of Nb3B2 at 1,200 °C at the B/Nb interface. At lower temperatures the high oxygen content in the boron layer is supposed to hinder the formation of borides due to formation of glass-like boron oxides. In NH3 several niobium nitrides are formed but no boride phases. Here again the reactivity of boron with niobium is suppressed by the high oxygen content and boron oxide formation. During annealing of the B/Nb2N bilayers no borides were formed indicating that well-formed Nb2N is an effective diffusion barrier for B.
Keywords: Niobium boride; Nitride; Rapid thermal processing (RTP); X-ray diffraction (XRD); SIMS depth profile

Surface analysis of different boundary layers on steel discs formed in a lubricated tribocontact during laboratory test compared with field application by Astrid Stadler; Josef Brenner; Andreas Pauschitz; Josef Wendrinsky; Andreas Schindel; Gerhard E. Nauer (1527-1535).
The aim of this study was to gain knowledge of tribological layers on a steel disc. This steel disc has been used in a transmission system in the field. ATF-oil (automatic transmission fluid), which contains different surface-adhering additives, serves as a lubricant and as a cooling fluid in the tribological system. Over time, the tribological characteristics of the system changed. Special interest in this study lies in the modifications of the steel surface. The field-used disc was therefore compared with model tested discs. In a disc-on-disc tribometer, all samples are tested for a short period of time to evaluate the actual condition of the system regarding friction behaviour. Analysis and characterisation of surfaces and layer formation were carried out with, among other techniques, 3D topography, SEM / EDX, AFM, XPS, and ToF-SIMS. Results indicate that in some regions a complex composed tribological layer is formed. Differences were detected between the steel discs used in the field and the model discs. This indicates the problem of evaluation of long-term behaviour exclusively by use of short or accelerated model experiments. A combination of carefully selected and sophisticated analytical methods is necessary to trace small changes of the system. Figure Atomic force measurement of field used and model tested “new” friction discs
Keywords: Tribology; Surface analysis; Additives; Lubricated tribo-contact; Tribo-layer

TOF-SIMS investigations on thermally treated copper–molybdenum films on a carbon substrate by Stefan Puchner; Herbert Hutter; Christoph Eisenmenger-Sittner; M. Kiniger (1537-1541).
Metal-matrix composites are made of materials with different physical and chemical properties. It is possible to change the mechanical, thermal and electrical properties by variation of the mass ratio of the components; therefore, metal-matrix composites have great value for industrial and technological applications. Copper–carbon composites have a good chance to be used as heat sinks for electronic components, which can be explained by their high thermal conductivity, low density and an adjustable coefficient of thermal expansion. On the other hand, the mechanical adhesion of copper and carbon is extremely weak because of their immiscibility and weak chemical interactions. In order to compensate for the low wettability of carbon by copper, a thin molybdenum intermediate layer is used as an adhesion promoter. In this work a time of flight secondary ion mass spectrometry technique was primarily used to detect the carbide formation in the molybdenum and copper layers, depending on different temperature conditions during sputter deposition and annealing afterwards. The CuMo layers were deposited by magnetron sputtering. The adhesion of the samples was determined by a destructive pull-off test. We found that heat treatment mainly modifies the carbide formation in the molybdenum and copper layers.
Keywords: Time of flight secondary ion mass spectrometry; CuMo films; Adhesion; Metal-matrix composites (MMC); Carbide formation

TOF-SIMS investigations on weathered silver surfaces by J. Schnöller; R. Wiesinger; C. Kleber; U. Hilfrich; M. Schreiner; H. Hutter (1543-1549).
Silver-coated quartz crystal microbalance (QCM) disks were treated under different environmental conditions (including changes in parameters such as relative humidity (%RH) and SO2/H2S content) in atmospheres of synthetic air and pure N2 for 24 h in a weathering chamber. The corroded surfaces were subjected to depth profiling by a time of flight (TOF) secondary ion mass spectrometry (SIMS) instrument, equipped with a Bi+ analysis gun and Cs+ sputter gun. The evaluation of the in-depth distribution of several elements and species provides evidence for the formation of a corrosion layer containing Ag2SO3, even in the absence of oxidizing agents, such as H2O2 or NO2. Furthermore it could be elucidated that the thickness of the formed Ag2SO3 layer does not depend on the SO2 concentration but rather on the humidity and oxygen content of the ambient atmosphere. In weathering experiments in atmospheres composed of synthetic air, humidity, and H2S, the presence of different oxygen species (surface and bulk) and silver sulfide could be detected by TOF-SIMS depth profiling experiments. The obtained results for both acidifying gases are in good correlation with the corresponding tapping mode atomic force microscopy (TM-AFM) investigations and in situ QCM measurements.
Keywords: TOF-SIMS; Depth profiling; QCM; TM-AFM; Weathering; Corrosion

There is a critical need for a rapid and sensitive means of detecting viruses. Recent reports from our laboratory have shown that surface-enhanced Raman spectroscopy (SERS) can meet these needs. In this study, SERS was used to obtain the Raman spectra of respiratory syncytial virus (RSV) strains A/Long, B1, and A2. SERS-active substrates composed of silver nanorods were fabricated using an oblique angle vapor deposition method. The SERS spectra obtained for each virus were shown to posses a high degree of reproducibility. Based on their intrinsic SERS spectra, the four virus strains were readily detected and classified using the multivariate statistical methods principal component analysis (PCA) and hierarchical cluster analysis (HCA). The chemometric results show that PCA is able to separate the three virus strains unambiguously, whereas the HCA method was able to readily distinguish an A2 strain-related G gene mutant virus (ΔG) from the A2 strain. The results described here demonstrate that SERS, in combination with multivariate statistical methods, can be utilized as a highly sensitive and rapid viral identification and classification method.
Keywords: Virus; SERS; Detection; RSV; Multivariate statistics; Nanorod

Self-assembled monolayers as a base for immunofunctionalisation: unequal performance for protein and bacteria detection by Eva Baldrich; Olivier Laczka; F. Javier del Campo; Francesc Xavier Muñoz (1557-1562).
Biosensor development strongly depends on the optimisation of surface functionalisation strategies. When gold surfaces are considered, immunofunctionalisation by modification of self-assembled monolayers (SAMs) is one of the preferred approaches. In this respect, SAM-based antibody (Ab) incorporation has shown better performance than Ab physisorption for the detection of proteins and small targets. Reports on bacteria detection are less frequent. In this work, we assess the performance of various SAM-based gold immunofunctionalisation strategies, currently applied to protein detection, in the field of bacteria determination. We present the results for Ab chemical conjugation on mercaptopropanoic acid and mercaptoundecanoic acid SAMs, as well as on a dextranized cysteamine SAM. All the modified surfaces studied were shown to be appropriate for the direct detection of an enzyme-labelled protein, but none succeeded in detecting a bacterial target in a sandwich assay format. Conversely, gold functionalised by Ab physisorption allowed E. coli detection when a sandwich enzyme-linked assay was carried out. The implications of bacteria size and wall complexity are discussed. These results indicate that immunofunctionalisation strategies appropriate for protein detection are not necessarily transferable to work with more complex targets such as bacteria. In this respect, Ab physisorption appears to be a suitable alternative to SAM-based gold functionalisation for bacteria detection.
Keywords: Surface immunofunctionalisation; Self-assembled monolayer; Antibody conjugation; Bacteria detection

Home-built integrated microarray system (IMAS). A three-laser confocal fluorescence scanner coupled with a microarray printer by Sotirios S. Tragoulias; Pierre J. Obeid; Ioannis E. Tataridis; Theodore K. Christopoulos (1563-1573).
Microarray technology covers the urgent need to exploit the accumulated genetic information from large-scale sequencing projects and facilitate investigations on a genome-wide scale. Although most applications focus on DNA microarrays, the technology has expanded to microarrays of proteins, peptides, carbohydrates, and small molecules aiming either at detection/quantification of biomolecules or investigation of biomolecular interactions in a massively parallel manner. Microarray experiments require two specialized instruments: An arrayer (or printer), for construction of microarrays, and a readout instrument (scanner). We have designed, constructed, and characterized the first integrated microarray system (IMAS) that combines the functions of a microarrayer and a three-laser confocal fluorescence scanner into a single instrument and provides excellent flexibility for the researcher. The three-axis robotic system that moves the printing head carrying multiple pins for arraying is also used for moving the microarray slide in front of a stationary optical system during scanning. Since the translation stages are the most expensive and crucial components of microarray printers and scanners, the proposed design reduces considerably the cost of the instrument and enhances remarkably its operative flexibility. Experiments were carried out at resolutions of 2.5, 5, 10, and 20 μm. The scanner detects 0.128 nmol L−1 carboxyfluorescein (spots with diameters of 70 μm) corresponding to 1.8 molecules μm−2. The linear range extends over 3.5 orders of magnitude (R 2 = 0.997) and the dynamic range covers almost five orders of magnitude. DNA microarray model experiments were carried out, including staining with SYBR Green I and hybridization with oligonucleotides labeled with the fluorescent dyes Alexa 488, Alexa 594, and Alexa 633. Figure Lay-out of the home-built integrated microarray system (IMAS). For the first time, the functions of a microarrayer (printer) and a three-laser confocal fluorescence scanner are combined into a single instrument. The three-axis robotic system that moves the printing head for arraying is also used to move the microarray slide in front of a stationary optical system during scanning.
Keywords: Bioanalytical methods; Microarrays; Home-built integrated system

We report the multiplexed, simultaneous analysis of antigen–antibody interactions that involve human immunoglobulin G (IgG) on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved by simultaneously monitoring protein–protein interactions under identical conditions. Antibody–antigen binding affinities towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement of protein–protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant molecules. Figure Microchannel SPR imaging for protein–protein interactions
Keywords: Surface plasmon resonance; SPR imaging; Human immunoglobulin G; Protein–protein interaction

Quantification of the trace element content of subcellular compartments is a challenging task because of the lack of analytical quantitative techniques with adequate spatial resolution and sensitivity. Ion beam micro-analysis, using MeV protons or alpha particles, offers a unique combination of analytical methods that can be used with micrometric resolution for the determination of chemical element distributions. This work illustrates how the association of three ion beam analytical methods, PIXE (particle induced X-ray emission), BS (backscattering spectrometry), and STIM (scanning transmission ion spectrometry), allows quantitative determination of the trace element content of single cells. PIXE is used for trace element detection while BS enables beam-current normalization, and STIM local mass determination. These methods were applied to freeze-dried cells, following a specific cryogenic protocol for sample preparation which preserves biological structures and chemical distributions in the cells. We investigated how iron accumulates into dopaminergic cells cultured in vitro. We found that the iron content increases in dopaminergic cells exposed to an excess iron, with marked accumulation within distal ends, suggesting interaction between iron and dopamine within neurotransmitter vesicles. Increased iron content of dopaminergic neurons is suspected to promote neurodegeneration in Parkinson’s disease.
Keywords: Ion beam analysis; PIXE; RBS; Trace element; Cell imaging

Binding of hairpin polyamides to DNA studied by fluorescence correlation spectroscopy for DNA nanoarchitectures by Chayan K. Nandi; Partha P. Parui; Thorsten L. Schmidt; Alexander Heckel; Bernhard Brutschy (1595-1603).
We have recently constructed a “DNA strut” consisting of two DNA-binding hairpin polyamides of Dervan-type connected via a long flexible linker and were able to show that this strut can be used to sequence-selectively connect DNA helices. This approach provides a second structural element (besides the Watson–Crick base pairing) for the assembly of higher-order DNA nanoarchitectures from smaller DNA building blocks. Since none of the existing analytical techniques for studying this kind of system were found suitable for detection and quantification of the formation of the resulting complexes, we chose fluorescence correlation spectroscopy (FCS). In the present study we show that FCS allowed us in a versatile and fast way to investigate the binding of Dervan polyamides to DNA. In particular it also shows its power in the quantitative detection of the formation of multimeric complexes and the in investigation of binding under nonphysiological conditions.
Keywords: Fluorescence correlation spectroscopy; DNA-binding polyamides; DNA nanoarchitectures; Single molecule spectroscopy; Multimeric complexes

In recent years, room temperature ionic liquids (RTILs) have proven to be of great interest to analytical chemists. One important development is the use of RTILs as highly thermally stable GLC stationary phases. To date, nearly all of the RTIL stationary phases have been nitrogen-based (ammonium, pyrrolidinium, imidazolium, etc.). In this work, eight new monocationic and three new dicationic phosphonium-based RTILs are used as gas–liquid chromatography (GLC) stationary phases. Inverse gas chromatography (GC) analyses are used to study the solvation properties of the phosphonium RTILs through a linear solvation energy model. This model describes the multiple solvation interactions that the phosphonium RTILs can undergo and is useful in understanding their properties. In addition, the phosphonium-based stationary phases are used to separate complex analyte mixtures by GLC. Results show that the small differences in the solvent properties of the phosphonium ILs compared with ammonium-based ILs will allow for different and unique separation selectivities. Also, the phosphonium-based stationary phases tend to be more thermally stable than nitrogen-based ILs, which is an advantage in many GC applications.
Keywords: Phosphonium; Ionic liquids; Characterization; Gas chromatography; Stationary phases; Thermal stability

To determine sulphamethazine (SMZ) residues in edible animal foods (pig muscle, chicken muscle, egg, fish, milk and liver), a competitive direct enzyme-linked immunosorbent assay (ELISA) and a colloidal gold immunoassay were established. The limits of detection of the ELISA and the colloidal gold immunoassay were 0.02 and 0.5 μg kg−1, respectively. The specificity of the ELISA developed to the SMZ was high according to the results of cross-reactivity testing with 14 kinds of sulphonamides. To obtain a more sensitive immunoassay, buffer solution (30 mmol L−1 phosphate-buffered saline with 0.05% Tween 20, pH 8.5) was optimized through the whole test procedure. A simple and efficient extraction method for the rapid detection of SMZ residues in foods was developed, with recoveries between 74 and 117.5%. Matrix effects can be avoided by 1:10 dilution of pig muscle, chicken muscle, egg, fish, milk and liver with optimal buffer. The detection limit of SMZ was 5 μg kg−1 in liver and 2 μg kg−1 in the other five samples. For the validation of the ELISA tests, sample extracts were analysed by ELISA and high-performance liquid chromatography. The results obtained by these two methods showed a good correlation (r 2) which was greater than 0.9. The colloidal gold immunoassay presented in this assay was successfully applied to determine SMZ in pig muscle, milk and fish below or equal to the maximum residue level (20 μg kg−1).
Keywords: Sulphamethazine; ELISA; Colloidal gold immunoassay; Food; Matrix effect

Identification of five Listeria species based on infrared spectra (FTIR) using macrosamples is superior to a microsample approach by Cecilia A. Rebuffo-Scheer; Jochen Dietrich; Mareike Wenning; Siegfried Scherer (1629-1635).
Microorganisms can be identified using both macrosamples and microsamples based on infrared spectra (FTIR). This work compares the identification of the five closely related Listeria species L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri, and L. welshimeri using both methods. The overall identification success for 25 strains was 92.8% for the former and 79.2% for the latter methods, respectively. The worst performances of the microsample method were obtained for L. innocua, L. ivanovii, and L. monocytogenes, while L. seeligeri and L. welshimeri did not show significant differences between the techniques. Identification success was mainly influenced by the age of the cells and the spatial heterogeneity of the microcolonies, as analyzed by the microsample method. Spectra of Listeria cells near the stationary phase exhibited more species-specific markers and thus allowed for better discrimination than spectra of growing cells. Furthermore, the heterogeneity of cell composition at different locations in microcolonies of L. innocua, L. ivanovii and L. monocytogenes resulted in limited discrimination success of the microsample method. We conclude that, at least in the case of Listeria, the macrosample method is superior to the microsample method, although the latter is the faster technique.
Keywords: Listeria monocytogenes ; Listeria species; FTIR macrosample method; FTIR microsample method; Species identification

Optimization of a rapid capillary electrophoresis ESI-IT tandem mass spectrometry method for the analysis of short-chain carnitines in human plasma by Claudia Desiderio; Antonella De Rossi; Rosanna Inzitari; Angelo Mancinelli; Diana Valeria Rossetti; Massimo Castagnola; Irene Messana (1637-1644).
A capillary electrophoresis (CE) method coupled to electrospray ionization ion trap tandem mass spectrometry (ESI-IT-MS/MS) is described for the rapid analysis of carnitine, acetylcarnitine, and propionylcarnitine in human plasma. Optimization of the procedure was achieved by a reduced sample pretreatment and after examining several physicochemical parameters that influence both the CE separation and the MS analytes detection. The analysis of total carnitine in human plasma after hydrolysis of short-chain metabolites is also shown. The analysis of carnitine and metabolites was obtained in less than 10 min using a 200 mM ammonium formate buffer, pH 2.5, with high sensitivity and specificity using the MS detection in product ion scan mode. The method was tested for quantitative recovery using dialyzed human plasma as matrix and showed linearity in the concentrations ranges 20–160, 1–32, and 0.25–8 μM for carnitine, acetylcarnitine, and propionylcarnitine with (squared) correlation coefficients of 0.9984, 0.9995, and 0.9991, respectively. The intraday and intermediate analysis repeatability and accuracy are within 15% of relative standard deviation (RSD) at low, medium, and high concentration and within/or slight exceeding 20% at the lower limit of quantitation (LLOQ). The method is sensitive for determining carnitine and its metabolites in human plasma with high specificity.
Keywords: Capillary electrophoresis; Mass spectrometry; Carnitines; Biological fluids; Sample preparation

A novel on-column sequential preconcentration method based on the combination of field-amplified sample injection induced by acetonitrile and pseudo isotachophoresis (ITP)–acid stacking is developed for simply but efficiently concentrating alkaloid cations in a high-salt sample matrix in capillary electrophoresis. Acetonitrile (70%) added to a sample solution with a high-salt sample matrix not only induces field-amplified sample stacking by decreasing conductivity but also acts as a termination reagent in the succeeding pseudo ITP. After sample injection had been completed, a plug of H+ was injected electrokinetically and a neutralization reaction between H+ and tartrate from the buffer solution produced a low conductivity zone, in which the injected analyte cations were further concentrated. With the sequential preconcentration method, a 3 orders of magnitude detection sensitivity (1,400-fold) increase could be observed compared with the conventional electrokinetic injection method, without compromising separation efficiency and peak shape, and detection limits of 0.1 ng/mL for myosmine and 0.3 ng/mL for anabasine with the conditions selected were achieved. The calibration curves demonstrated good linearity in the concentration ranges 1.3–600 ng/mL for myosmine and 4.9–900 ng/mL for anabasine, respectively. The proposed method has been used to analyze successfully trace alkaloids in cigarette samples. Figure Sequential preconcentration processes: a sample injection; b introduction of HCl; c capillary zone electrophoresis separation. A tartrate, white circles acetonitrile, black circles Na+, sample zone, myosmine, anabasine
Keywords: Capillary electrophoresis; Field-amplified sample injection; Acid stacking; Pseudo isotachophoresis; Tobacco alkaloids; High-salt sample

Neutron activation analysis and X-ray Rayleigh and Raman scattering of hair and nail clippings as noninvasive bioindicators for Cu liver status in Labrador Retrievers by Peter Bode; Maria Izabel Maretti Silveira Bueno; Gisele G. Bortoleto; Gaby Hoffmann; Ted S. G. A. M. van den Ingh; Jan Rothuizen (1653-1658).
The heritability of chronic hepatitis in the Labrador Retriever is studied with the aim of identifying the related gene mutation. Identification of cases and controls is largely based on instrumental neutron activation analysis (INAA) Cu determination in liver biopsies. The burden for these companion animals may be reduced if nail clippings and hair (fur) could serve as a noninvasive indicator for the hepatic Cu concentrations. No correlation was found between hepatic Cu concentrations and Cu concentrations in hair and nail samples. However, hair and nail samples were also analyzed by X-ray tube excitation, taking advantage of the X-ray Compton, Rayleigh, and Raman scattering which reflects the organic components such as the type of melanin. Principal component analysis provided first indications that some differentiation between healthy and sick dogs could indeed be obtained from hair and nail analysis. Figure Principal component analysis of scattered region of x-ray fluorescence spectra of Labrador dog nails, demonstrating the differentiation towards dogs with high and low Cu liver levels (respectively positive and negative PC2 values) reflecting hepatitis, as well as gender (PC1: negative values for female and positive values for males)
Keywords: Neutron activation analysis; X-ray spectroscopy; Chemometrics; Trace elements; Biological samples

Structure and composition of the nacre–prisms transition in the shell of Pinctada margaritifera (Mollusca, Bivalvia) by Yannicke Dauphin; Alexander D. Ball; Marine Cotte; Jean-Pierre Cuif; Anders Meibom; Murielle Salomé; Jean Susini; C. Terry Williams (1659-1669).
A microstructural, mineralogical, and chemical study of the nacre–prisms boundary in the shells of Pinctada margaritifera shows that this boundary is not an abrupt transition, but that there exists a distinct fibrous layer with clear topographic structures and evidence of growth lines. A three-step biomineralization process is proposed that involves changes in the chemical and biochemical composition of the last growth increments of the calcite prisms, formation of the fibrous layer, and development of regular tablets in the nacreous layer.
Keywords: Biomineralization; Pinctada margaritifera ; X-ray absorption near-edge structure; Fourier transform IR; Laser confocal microscopy; Atomic force microscope

In recent years, bamboo charcoal, a new kind of material with special microporous and biological characteristics, has attracted great attention in many application fields. In this paper, the potential of bamboo charcoal to act as a solid-phase extraction (SPE) adsorbent for the enrichment of the environmental pollutant perfluorooctanoic acid, which is one of the newest types of persistent organic pollutants in the environment, has been investigated. Important factors that may influence the enrichment efficiency—such as the eluent and its volume, the flow rate of the sample, the pH of the sample and the sample volume—were investigated and optimized in detail. Under the optimum conditions, the limit of detection for PFOA was 0.2 ng L−1. The experimental results indicated that this approach gives good linearity (R 2 = 0.9995) over the range 1–1000 ng L−1 and good reproducibility, with a relative standard deviation of 4.0% (n = 5). The proposed method has been applied to the analysis of real water samples, and satisfactory results were obtained. The average spiked recoveries were in the range 79.5∼118.3 %. All of the results indicate that the proposed method could be used for the determination of PFOA at ultratrace levels in water samples.
Keywords: Perfluorooctanoic acid; Bamboo charcoal; Solid-phase extraction; High-performance liquid chromatography–mass spectrometry