Analytical and Bioanalytical Chemistry (v.396, #1)

Analytical tools for the nanoworld by Renato Zenobi (1-1).
has been Professor of Analytical Chemistry in the Organic Chemistry Laboratory of ETH Zurich since 1995. He received an M.S. degree from ETH Zurich in 1986 and a Ph.D. degree from Stanford University in the USA in 1990. He had two postdoctoral appointments at the University of Pittsburgh (1990–1991) and at the University of Michigan (1991). Renato Zenobi’s research interests include laser-based analytical chemistry, electrospray and laser-assisted mass spectrometry, laser-surface interactions, and near-field optical microscopy and spectroscopy. He has received numerous awards for his scientific work, including the Thomas Hirschfeld Award, the Ruzicka Prize, the Heinrich Emanuel Merck Prize, the Theophilus Redwood Lectureship, and the Michael Widmer Award.

Microcavities: tailoring the optical properties of single quantum emitters by Sebastian Bär; Alexey Chizhik; Raphael Gutbrod; Frank Schleifenbaum; Anna Chizhik; Alfred J. Meixner (3-14).
We present a general review of different microresonator structures and how they can be used in future device applications in modern analytical methods by tailoring the optical properties of single quantum emitters. The main emphasis is on the tunable λ/2-Fabry–Perot-type microresonator which we used to obtain the results presented in this article. By varying the mirror distance the local mode structure of the electromagnetic field is altered and thus the radiative coupling of fluorescent single quantum emitters embedded inside the resonator to that field is changed, too. As a result a modification of the optical properties of these quantum emitters can be observed. We present experimental as well as theoretical results illustrating this effect. Furthermore, the developed resonator can be used to determine the longitudinal position of embedded emitters with an accuracy of λ/60 by analyzing the excitation patterns of nano-sized fluorescent polymer spheres after excitation with a radially polarized doughnut mode laser beam. Finally, we will apply this resonator to a biological system and demonstrate the modification of Förster resonant energy transfer (FRET) efficiency by inhibiting the excited state energy transfer from the donor to the acceptor chromophore of a single DsRed protein. Figure Effect of a microresonator on single quantum emitters (from left to right): PI molecule or DsRed protein invesitigated in a microresonator with resulting exciation patterns of the PI molecule after exciation with a radially polarized laser beam or the cavity-controlled emisison spectrum of DSRed in comparison with its free space spectrum (hatched). The background shows the Newton rings of the microrsonator.
Keywords: Microresonator; Single molecule spectroscopy; Confocal microscopy; Autofluorescent proteins; Higher-order laser modes; FRET

Inorganic mass spectrometry as a tool for characterisation at the nanoscale by Beatriz Fernández; Jose Manuel Costa; Rosario Pereiro; Alfredo Sanz-Medel (15-29).
Inorganic mass spectrometry techniques may offer great potential for the characterisation at the nanoscale, because they provide unique elemental information of great value for a better understanding of processes occurring at nanometre-length dimensions. Two main groups of techniques are reviewed: those allowing direct solid analysis with spatial resolution capabilities, i.e. lateral (imaging) and/or in-depth profile, and those for the analysis of liquids containing colloids. In this context, the present capabilities of widespread elemental mass spectrometry techniques such as laser ablation coupled with inductively coupled plasma mass spectrometry (ICP-MS), glow discharge mass spectrometry and secondary ion/neutral mass spectrometry are described and compared through selected examples from various scientific fields. On the other hand, approaches for the characterisation (i.e. size, composition, presence of impurities, etc.) of colloidal solutions containing nanoparticles by the well-established ICP-MS technique are described. In this latter case, the capabilities derived from the on-line coupling of separation techniques such as field-flow fractionation and liquid chromatography with ICP-MS are also assessed. Finally, appealing trends using ICP-MS for bioassays with biomolecules labelled with nanoparticles are delineated. Figure Inorganic mass spectrometry: an emerging tool for nanotechnology
Keywords: Inorganic mass spectrometry; Nanoparticles; Imaging; Spatial resolution; Depth profile analysis

Near-field scanning optical microscopy: a tool for nanometric exploration of biological membranes by Nicholas E. Dickenson; Kevin P. Armendariz; Heath A. Huckabay; Philip W. Livanec; Robert C. Dunn (31-43).
Near-field scanning optical microscopy (NSOM) is an emerging optical technique that enables simultaneous high-resolution fluorescence and topography measurements. Here we discuss selected applications of NSOM to biological systems that help illustrate the utility of its high spatial resolution and simultaneous collection of both fluorescence and topography. For the biological sciences, these attributes seem particularly well suited for addressing ongoing issues in membrane organization, such as those regarding lipid rafts, and protein–protein interactions. Here we highlight a few NSOM measurements on model membranes, isolated biological membranes, and cultured cells that help illustrate some of these capabilities. We finish by highlighting nontraditional applications of NSOM that take advantage of the small probe to create nanometric sensors or new modes of imaging.
Keywords: Near-field scanning optical microscopy; Fluorescence; Membranes; Nuclear pores; Single molecule

Development of tip-enhanced optical spectroscopy for biological applications: a review by Alistair P. D. Elfick; Andrew R. Downes; Rabah Mouras (45-52).
Tip-enhanced optical spectroscopy is an approach that holds a good deal of promise for the nanoscale characterisation of matter. Tip-enhanced Raman spectroscopy (TERS) has been demonstrated on a variety of samples: inorganic, organic and biological. Imaging using TERS has been shown for carbon nanotubes due to their high scattering efficiency. There are a number of compelling motivations to consider alternative approaches for biological samples; most importantly, the potential for heat damage of biomolecules and long acquisition times. These issues may be addressed through the development of tip-enhanced coherent anti-Stokes Raman scattering microscopy.
Keywords: TERS; Raman; Near-field; Biological samples; tip-enhanced CARS

Nanostructured optical fibre arrays for high-density biochemical sensing and remote imaging by F. Deiss; N. Sojic; D. J. White; P. R. Stoddart (53-71).
Optical fibre bundles usually comprise a few thousand to tens of thousands of individually clad glass optical fibres. The ordered arrangement of the fibres enables coherent transmission of an image through the bundle and therefore enables analysis and viewing in remote locations. In fused bundles, this architecture has also been used to fabricate arrays of various micro to nano-scale surface structures (micro/nanowells, nanotips, triangles, etc.) over relatively large areas. These surface structures have been used to obtain new optical and analytical capabilities. Indeed, the imaging bundle can be thought of as a “starting material” that can be sculpted by a combination of fibre drawing and selective wet-chemical etching processes. A large variety of bioanalytical applications have thus been developed, ranging from nano-optics to DNA nanoarrays. For instance, nanostructured optical surfaces with intrinsic light-guiding properties have been exploited as surface-enhanced Raman scattering (SERS) platforms and as near-field probe arrays. They have also been productively associated with electrochemistry to fabricate arrays of transparent nanoelectrodes with electrochemiluminescent imaging properties. The confined geometry of the wells has been loaded with biosensing materials and used as femtolitre-sized vessels to detect single molecules. This review describes the fabrication of high-density nanostructured optical fibre arrays and summarizes the large range of optical and bioanalytical applications that have been developed, reflecting the versatility of this ordered light-guiding platform.
Keywords: Nanosensor array; Biosensor; Imaging; SERS; Electrochemistry; Single molecule detection; Optical fibre sensors

Single-walled carbon nanotube as an effective quencher by Zhi Zhu; Ronghua Yang; Mingxu You; Xiaoling Zhang; Yanrong Wu; Weihong Tan (73-83).
Over the past few years, single-walled carbon nanotubes (SWNTs) have been the focus of intense research motivated by their unique physical and chemical properties. This review specifically summarizes recent progress in the development of fluorescence biosensors that integrate the quenching property of SWNTs and the recognition property of functional nucleic acids. SWNTs are substantially different from organic quenchers, showing superior quenching efficiency for a variety of fluorophores, with low background and high signal-to-noise ratio, as well as other advantages derived from the nanomaterial itself. As the second key component of biosensors, functional nucleic acids can bind to either their complementary DNA or a target molecule with the ability to recognize a broad range of targets from metal ions to organic molecules, proteins, and even live cells. By taking advantage of the strengths and properties of both SWNTs and nucleic acid based aptamers, a series of fluorescence biosensors have been designed and fabricated for the detection of a broad range of analytes with high selectivity and sensitivity.
Keywords: Single-walled carbon nanotubes; Quencher; Biosensor; Molecular beacon; Aptamer; Singlet oxygen generation

A new SIMS paradigm for 2D and 3D molecular imaging of bio-systems by John S. Fletcher; John C. Vickerman (85-104).
With the implementation of focused primary ion beams, secondary ion mass spectrometry (SIMS) has become a significant technique in the rapidly emerging field of mass spectral imaging in the biological sciences. Liquid metal ion guns (LMIG) offered the prospect of sub-100 nm spatial resolution, however this aspiration has yet to be reached for molecular imaging. This brief review shows that using LMIG the limitations of the static limit and low ionization probability will restrict useful imaging to around 2 μm spatial resolution with high-yield molecules. The only prospect of going beyond this in the absence of factors of 100 increase in ionization probability is to use polyatomic ion beams such as C 60 + , for which bombardment induced damage is low. In these cases sub-micron imaging becomes possible, using voxels together with molecular depth profiling and 3D imaging. The discussion shows that conventional ToF-SIMS instrumentation then becomes a limitation in that the pulsed ion beam has a very low duty cycle which results in inordinately long analysis times, and pulsing the beam means that high-mass resolution and high spatial resolution are mutually incompatible. New instrumental configurations are described that allow the use of a dc ion beam and separate the mass spectrometry for the ion formation process. Early results from these instruments suggest that sub-micron analysis and imaging with high mass resolution and good ion yields are now realizable, although the low ion yield issue still needs to be solved.
Keywords: SIMS; Imaging mass spectrometry; Depth profiling; 2D imaging; 3D imaging

Molecular sputter depth profiling using carbon cluster beams by Andreas Wucher; Nicholas Winograd (105-114).
Sputter depth profiling of organic films while maintaining the molecular integrity of the sample has long been deemed impossible because of the accumulation of ion bombardment-induced chemical damage. Only recently, it was found that this problem can be greatly reduced if cluster ion beams are used for sputter erosion. For organic samples, carbon cluster ions appear to be particularly well suited for such a task. Analysis of available data reveals that a projectile appears to be more effective as the number of carbon atoms in the cluster is increased, leaving fullerene ions as the most promising candidates to date. Using a commercially available, highly focused C 60 q+ cluster ion beam, we demonstrate the versatility of the technique for depth profiling various organic films deposited on a silicon substrate and elucidate the dependence of the results on properties such as projectile ion impact energy and angle, and sample temperature. Moreover, examples are shown where the technique is applied to organic multilayer structures in order to investigate the depth resolution across film-film interfaces. These model experiments allow collection of valuable information on how cluster impact molecular depth profiling works and how to understand and optimize the depth resolution achieved using this technique.
Keywords: Molecular depth profiling; Cluster SIMS; Carbon clusters; Cluster ion beams

The fundamental parameters and limitations that determine the signal strength in tip-enhanced Raman scattering (TERS) are discussed. A semiquantitative analysis of the Raman signal expected in different experimental geometries and with different sample systems is presented, taking into account experimental parameters including Fresnel factor, numerical aperture of the illumination and collection optics, detection efficiency, and the Raman scattering cross section of the material. A top/side-on illumination geometry is essential for the study of nontransparent samples. It can yield the highest signal levels when strong tip–sample coupling using a metallic substrate provides large field enhancement. In contrast, axial/through-sample illumination is limited to transparent sample materials. Although conceptually simpler in experimental implementation and despite high numerical aperture signal collection efficiency, signals are generally weaker due to limited field enhancement. Crystalline solids with small Raman cross sections and dense molecular/biological systems with unavoidable far-field background provide the biggest challenge for TERS analysis yet at the same time hold the most exciting outstanding scientific questions TERS has the potential to answer. Figure 3 Excitation and emission sequence in tip-enhanced Raman scattering. The signal intensity can be estimated for a given experimental layout considering numerical aperture, Raman scattering cross-section, and plasmonic field enhancement.
Keywords: TERS; Raman scattering cross section; Sensitivity; Signal levels

Time-resolved confocal fluorescence microscopy of trinitrobenzene-responsive organic nanofibers by Carlo Giansante; Alexandre G. L. Olive; Christian Schäfer; Guillaume Raffy; André Del Guerzo (125-131).
Time-resolved confocal fluorescence microscopy is used to image and analyze quantitatively the influence of 1,3,5-trinitrobenzene on the fluorescence of organic nanofibers. These nanofibers are formed by self-assembly of 2,3-didecyloxyanthracene in methanol or from solutions drop-casted onto glass surfaces. Amplification of the fluorescence quenching emerges in the nanofibers as compared to the constituting monomer thus leading to efficient detection of nanomolar concentrations of TNB. The emission of dry nanofibers on glass is also efficiently quenched by vapors of TNB.
Keywords: Confocal fluorescence microscopy; Fluorescence; Self-assembly; Nanofibers; Trinitrobenzene; Luminescence; Nanoparticles; Nanotechnology; Organic compounds; Trace organic compounds; Spectroscopy; Instrumentation

Atomic-force-controlled capillary electrophoretic nanoprinting of proteins by Yulia Lovsky; Aaron Lewis; Chaim Sukenik; Eli Grushka (133-138).
The general nanoprinting and nanoinjection of proteins on non-conducting or conducting substrates with a high degree of control both in terms of positional and timing accuracy is an important goal that could impact diverse fields from biotechnology (protein chips) to molecular electronics and from fundamental studies in cell biology to nanophotonics. In this paper, we combine capillary electrophoresis (CE), a separation method with considerable control of protein movement, with the unparalleled positional accuracy of an atomic force microscope (AFM). This combination provides the ability to electrophoretically or electroosmotically correlate the timing of protein migration with AFM control of the protein deposition at a high concentration in defined locations and highly confined volumes estimated to be 2 al. Electrical control of bovine serum albumin printing on standard protein-spotting glass substrates is demonstrated. For this advance, fountain pen nanolithography (FPN) that uses cantilevered glass-tapered capillaries is amended with the placement of electrodes on the nanopipette itself. This results in imposed voltages that are three orders of magnitude less than what is normally used in capillary electrophoresis. The development of atomic-force-controlled capillary electrophoretic printing (ACCEP) has the potential for electrophoretic separation, with high resolution, both in time and in space. The large voltage drop at the tip of the tapered nanopipettes allows for significant increases in concentration of protein in the small printed volumes. All of these attributes combine to suggest that this methodology should have a significant impact in science and technology. Figure Diagrammatic representation of the atomic-force-controlled electrophoretic nanoprinting (ACCEP) process, (on left). The combination of capillary electrophoresis with the unparalleled positional accuracy of an atomic force microscope allows for highly controlled nanoprinting of the fluorescently labelled protein bovine serum albumin on a non-conducting substrate (on right)
Keywords: AFM (atomic force microscopy); Capillary electrophoresis; Electrophoresis; Nanoparticles; Nanotechnology

Combining imaging ellipsometry and grazing incidence small angle X-ray scattering for in situ characterization of polymer nanostructures by Volker Körstgens; Johannes Wiedersich; Robert Meier; Jan Perlich; Stephan V. Roth; Rainer Gehrke; Peter Müller-Buschbaum (139-149).
A combination of microbeam grazing incidence small angle X-ray scattering (μGISAXS) and imaging ellipsometry is introduced as a new versatile tool for the characterization of nanostructures. μGISAXS provides a local lateral and depth-sensitive structural characterization, and imaging ellipsometry adds the position-sensitive determination of the three-dimensional morphology in terms of thickness, roughness, refractive index, and extinction coefficient. Together μGISAXS and imaging ellipsometry enable a complete characterization of structure and morphology. On the basis of an example of buildup of nanostructures from monodisperse colloidal polystyrene nanospheres on a rough solid support, the scope of this new combination is demonstrated. Roughness is introduced by a dewetting structure of a diblock copolymer film with one block being compatible with the colloidal nanoparticles and one block being incompatible. To demonstrate the potential for kinetic investigations, μGISAXS and imaging ellipsometry are applied to probe the drying process of an aqueous dispersion of nanospheres on such a type of rough substrate.
Keywords: Interface/surface analysis; Diffraction methods (low-energy electron diffraction|X-ray); Nanoparticles/nanotechnology; Microbeam grazing incidence small angle X-ray scattering; Imaging ellipsometry; Self-assembly

Mass spectrometry imaging of rat brain sections: nanomolar sensitivity with MALDI versus nanometer resolution by TOF–SIMS by Farida Benabdellah; Alexandre Seyer; Loïc Quinton; David Touboul; Alain Brunelle; Olivier Laprévote (151-162).
Mass spectrometry imaging is becoming a more and more widely used method for chemical mapping of organic and inorganic compounds from various surfaces, especially tissue sections. Two main different techniques are now available: matrix-assisted laser desorption/ionizaton, where the sample, preliminary coated by an organic matrix, is analyzed by a UV laser beam; and secondary ion mass spectrometry, for which the target is directly submitted to a focused ion beam. Both techniques revealed excellent performances for lipid mapping of tissue surfaces. This article will discuss similarities, differences, and specificities of ion images generated by these two techniques in terms of sample preparation, sensitivity, ultimate spatial resolution, and structural analysis.
Keywords: Mass spectrometry imaging; MALDI; TOF–SIMS; Lipid; Rat brain

Material ejection and redeposition following atmospheric pressure near-field laser ablation on molecular solids by Liang Zhu; Gerardo Gamez; Thomas A. Schmitz; Frank Krumeich; Renato Zenobi (163-172).
Near-field laser ablation (NF-LA) coupled with mass spectrometry (MS) is very promising for highly spatially resolved chemical analyses on various substrates at atmospheric pressure, for example, in materials and life science applications. Although nanoscale sample craters can be produced routinely, no molecular mass spectra of ablated material from craters of ≤1 µm diameter have ever been acquired by NF-LA MS at atmospheric pressure. Some of the pressing questions are thus how much of the ablated material is transported into the mass spectrometer and in what form. Therefore, material redeposition on the near-field tip’s surface from laser ablation of molecular solids was characterized with scanning electron microscopy. The crater profiles were studied by scanning probe microscopy. The results shown in this study demonstrate that there could be as much as 70% of the ablated material deposited on the near-field tip’s surface. The redeposited products were found to be confined to a height of ~50 µm, thus suggesting that most components inside near-field ablation plumes propagate about the same distance for both anthracene and tris(8-hydroxyquinolinato)aluminum. Nanoparticles ablated from craters of ≤1 µm diameter are clearly observed. Furthermore, observation of tips after ablation of an anthracene surface angled at 60° with respect to a horizontal surface shows that the direction of the near-field ablation plume is neither in the direction of the surface normal nor towards the axis of incident laser beam but deflected further away from surface normal. Figure Material redeposition on the near-field tip's surface from laser ablation of molecular solids was characterized with scanning electron microscopy.
Keywords: Near field; Laser ablation; Molecular solids; Atmospheric pressure; SEM

Optical far- and near-field femtosecond laser ablation of Si for nanoscale chemical analysis by Vassilia Zorba; Xianglei Mao; Richard E. Russo (173-180).
Extending spatial resolution in laser-based chemical analysis to the nanoscale becomes increasingly important as nanoscience and nanotechnology develop. Implementation of femtosecond laser pulses arises as a basic strategy for increasing resolution since it is associated with spatially localized material damage. In this work we study femtosecond laser far- and near-field processing of silicon (Si) at two distinct wavelengths (400 and 800 nm), for nanoscale chemical analysis. By tightly focusing femtosecond laser beams in the far-field, we were able to produce sub-micrometer craters. In order to further reduce the crater size, similar experiments were performed in the near-field through sub-wavelength apertures, resulting in the formation of sub-30-nm craters. Laser-induced breakdown spectroscopy (LIBS) was used for chemical analysis with a goal to identify the minimum crater size from which spectral emission could be measured. Emission from sub-micrometer craters (full width at half maximum) was possible, which are among the smallest ever reported for femtosecond LIBS.
Keywords: Laser ablation; LIBS; Silicon; Far-field; Near-field; Laser spectroscopy; Semiconductor materials

recently retired from Environment Canada, after managing a major analytical laboratory for 18 years. He is a graduate of the universities of London, UK, and Waterloo, Canada. He is a Fellow of the Chemical Institute of Canada, and is a member of the Advisory Board of Analytical and Bioanlaytical Chemistry.

Beryllium valence challenge by Juris Meija (185-186).

Long-term data archiving by David S. Moore (189-192).
is a Fellow of the American Physical Society (2004), received for breakthroughs in the use of nonlinear optical and ultrafast spectroscopies to understand the behavior of molecules under shock compression. He is presently Chair of the APS Topical Group on Shock Compression of Condensed Matter. He has been active in the IUPAC Analytical Chemistry Division since 1987, serving as Division President 2002–2003. He is presently a Research Scientist and Team Leader in the Shock and Detonation Physics Group at Los Alamos. His research team studies shock-induced physics and chemistry using ultrafast laser methods and develops new explosives detection methods, including dynamic detection using optimally shaped ultrafast lasers.

Searching for signals in the noise: metabolomics in chemical ecology by Emily K. Prince; Georg Pohnert (193-197).
Chemically mediated interactions between organisms influence ecosystem structure, making it crucial for ecologists to understand these interactions. Advances in chemical ecology have often been closely linked to advances in analytical chemistry techniques. One recent development is the use of metabolomics to address questions in chemical ecology. Although metabolomics has much to offer this field, it is not without drawbacks. Here we consider how metabolomics techniques can supplement the traditional bioassay-guided fractionation approach to chemical ecology. We focus on specific examples that illustrate the advantages that metabolomic methods can provide over other methods in order to understand chemically mediated interactions between organisms.
Keywords: Metabolic profiling; Metabolic fingerprinting; Chemical signaling; Brassicaceae; Caenorhabditis elegans

Specific enrichment methods for glycoproteome research by Lijuang Zhang; Haojie Lu; Pengyuan Yang (199-203).
Glycosylation is, by far, one of the most common and important post-translational modifications and becomes a target for proteomic research. A key challenge in glycoproteome research is the development of fast and effective enrichment strategies for high-throughput glycosylation analysis. Different kinds of glycan-capturing anchors have been developed and successfully applied to glyco-specific enrichment in large-scale glycosylation identification in the past few years. In this paper, we highlight several examples on various types of enrichment methods that have been utilized to specifically capture glycopeptides/glycoproteins for subsequent mass spectrometric analysis.
Keywords: Glycoproteome; Enrichment; Carbohydrate; Glycosylation

Diamond-related materials as potential new media in separation science by Pavel N. Nesterenko; Paul R. Haddad (205-211).
Recent progress in the development of various diamond-related materials (DRMs) has induced a strong interest in their use as a stationary phase in various separation techniques. DRMs meet many requirements for use as a stationary phase in chromatography, including excellent mechanical and chemical stability, high thermal stability, low chemical reactivity of the surface, and biocompatibility. The general physicochemical properties of diamond and the preparation of different types of DRMs are reviewed, and an overview is provided of current and possible future applications in solid-phase extraction and various separation technologies.
Keywords: Nanodiamonds; Ultradisperse diamonds; Chromatography; Solid-phase extraction

Unilateral NMR applied to the conservation of works of art by Eleonora Del Federico; Silvia A. Centeno; Cindie Kehlet; Penelope Currier; Denise Stockman; Alexej Jerschow (213-220).
In conventional NMR, samples from works of art in sizes above those considered acceptable in the field of art conservation would have to be removed to place them into the bore of large superconducting magnets. The portable permanent-magnet-based systems, by contrast, can be used in situ to study works of art, in a noninvasive manner. One of these portable NMR systems, NMR-MOUSE®, measures the information contained in one pixel in an NMR image from a region of about 1 cm2, which can be as thin as 2–3 µm. With such a high depth resolution, profiles through the structures of art objects can be measured to characterize the materials, the artists’ techniques, and the deterioration processes. A novel application of the technique to study a deterioration process and to follow up a conservation treatment is presented in which micrometer-thick oil stains on paper are differentiated and characterized. In this example, the spin–spin relaxation T 2 of the stain is correlated to the iodine number and to the degree of cross-linking of the oil, parameters that are crucial in choosing an appropriate conservation treatment to remove them. It is also shown that the variation of T 2 over the course of treatments with organic solvents can be used to monitor the progress of the conservation interventions. It is expected that unilateral NMR in combination with multivariate data analysis will fill a gap within the set of high-spatial-resolution techniques currently available for the noninvasive analysis of materials in works of art, where procedures to study the inorganic components are currently far more developed than those suitable for the study of the organic components.
Keywords: Unilateral NMR; Low-field NMR; Transverse relaxation; Oil stains; Art conservation

Ice chromatography: current progress and future developments by Tetsuo Okada; Yuiko Tasaki (221-227).
Ice chromatography, in which water-ice particles are employed as a chromatographic stationary phase, has proven an efficient technique for probing the solution/ice interface. The preparation of fine ice particles has allowed us to not only obtain higher-resolution separation but also investigate the molecular processes occurring on the ice surface in more detail. Chromatographic investigations have revealed that two or more hydrogen bonds are simultaneously formed between a solute and the dangling bonds on the ice surface when the solute gives measurable retention. Several compounds, including estrogens, amino acids, and acyclic polyethers, have been successfully separated by ice chromatography with a hexane-based mobile phase. In addition, this method effectively probes the surface melting of the ice stationary phase and the liquid phase that coexists with water ice at thermodynamic equilibrium. The thickness of the surface liquid layer and the size of the liquid phase that grows inside an ice particle have been evaluated. The perspectives of this method are also discussed. Figure Ice chromatographic separation of dyes in black ink
Keywords: Water ice; Liquid phase coexisting with ice; Separation; Interface; Quasi-liquid layer

Bioconjugated quantum dots as fluorescent probes for bioanalytical applications by Manuela F. Frasco; Nikos Chaniotakis (229-240).
Quantum dots (QDs) are inorganic semiconductor nanocrystals that have unique optoelectronic properties responsible for bringing together multidisciplinary research to impel their potential bioanalytical applications. In recent years, the many remarkable optical properties of QDs have been combined with the ability to make them increasingly biocompatible and specific to the target. With this great development, QDs hold particular promise as the next generation of fluorescent probes. This review describes the developments in functionalizing QDs making use of different bioconjugation and capping approaches. The progress offered by QDs is evidenced by examples on QD-based biosensing, biolabeling, and delivery of therapeutic agents. In the near future, QD technology still faces some challenges towards the envisioned broad bioanalytical purposes. Figure Bioanalytical applications of luminescent quantum dot-bioconjugates
Keywords: Quantum dots; Bioconjugation; Fluorescence; Biosensing; Biolabeling and imaging

The use of nanoparticles in electroanalysis: an updated review by Fallyn W. Campbell; Richard G. Compton (241-259).
The use of nanoparticles in electroanalysis is an area of research which is continually expanding. A wealth of research is available discussing the synthesis, characterization and application of nanoparticles. The unique properties of nanoparticulate materials (e.g. enhanced mass transport, high surface area, improved signal-to-noise ratio) can often be advantageous in electroanalytical techniques. The aim of this paper is to provide an updated overview of the work in this field. In this review we have concentrated on the advances with regards to silver, gold, platinum, palladium, ruthenium, copper and nickel. The synthesis, characterization and practical application of these materials are discussed. We have also identified the conditions under which each metal is likely to be stable, which is likely to be a useful tool for those practising in the field. Furthermore, we have provided a theoretical overview of advances in the theoretical modelling and simulation of nanoparticle behaviour.
Keywords: Nanoparticles; Electroanalysis; Simulation; Mass transport

Nano- and/or macrostructuring of electrode surfaces has recently emerged as a powerful method of improving the performances of electrochemical devices by enhancing both molecular accessibility and rapid mass transport via diffusion, by increasing the electroactive surface area in comparison to the geometric one, and/or by providing confinement platforms for hosting suitable reagents. This brief overview highlights how template technology offers advantages in terms of designing new types of porous electrodes—mostly based on thin films, and functionalized or not—and discusses their use in analytical chemistry via some recent examples from the literature on electrochemical sensors and biosensors. Figure
Keywords: Modified electrode; Thin film; Template; Self-assembly; Macroporous; Mesoporous; Metal; Ceramic; Organic–inorganic hybrid; Nanotechnology; Sensors and biosensors

Passive sampling as a tool for obtaining reliable analytical information in environmental quality monitoring by Bożena Zabiegała; Agata Kot-Wasik; Magdalena Urbanowicz; Jacek Namieśnik (273-296).
Passive sampling technology has been developing very quickly for the past 20 years, and is widely used for monitoring pollutants in different environments, for example air, water, and soil. It has many significant advantages, including simplicity, low cost, no need for expensive and complicated equipment, no power requirements, unattended operation, and the ability to produce accurate results. The present generation of passive samplers enables detection and analysis of bioavailable pollutants at low and very low concentrations and investigation of the environmental concentration of organic and inorganic pollutants not only on the local scale but also on continental and global scales. This review describes the current application of passive sampling techniques in environmental analysis and monitoring, under both equilibrium and non-equilibrium conditions.
Keywords: Passive sampling; Monitoring; Environmental quality studies; Water quality control; Air quality control

Relevance and use of capillary coatings in capillary electrophoresis–mass spectrometry by C. Huhn; R. Ramautar; M. Wuhrer; G. W. Somsen (297-314).
Over the last two decades, coupled capillary electrophoresis (CE)–mass spectrometry (MS) has developed into a generally accepted technique with a wide applicability. A growing number of CE-MS applications make use of capillaries where the internal wall is modified with surface coating agents. In CE-MS, capillary coatings are used to prevent analyte adsorption and to provide appropriate conditions for CE-MS interfacing. This paper gives an overview of the various capillary coating strategies used in CE-MS. The main attention is devoted to the way coatings can contribute to a proper CE-MS operation. The foremost capillary coating methods are discussed with emphasis on their compatibility with MS detection. The role of capillary coatings in the control of the electroosmotic flow and the consequences for CE-MS coupling are treated. Subsequently, an overview of reported applications of CE-MS employing different coating principles is presented. Selected examples are given to illustrate the usefulness of the coatings and the overall applicability of the CE-MS systems. It is concluded that capillary coatings can enhance the performance and stability of CE-MS systems, yielding a highly valuable and reproducible analytical tool.
Keywords: Capillary electrophoresis; Mass spectrometry; Capillary coatings; Review

Two-dimensional layered inorganic solids, such as cationic clays and layered double hydroxides (LDHs), also defined as anionic clays, have open structures which are favourable for interactions with enzymes and which intercalate redox mediators. This review aims to show the interest in clays and LDHs as suitable host matrices likely to immobilize enzymes onto electrode surfaces for biosensing applications. It is meant to provide an overview of the various types of electrochemical biosensors that have been developed with these 2D layered materials, along with significant advances over the last several years. The different biosensor configurations and their specific transduction procedures are discussed. Scheme of enzyme biosensors based on clay modified electrodes
Keywords: Clays; Layered double hydroxides; Enzymes; Modified electrodes; Biosensors

Update on analytical methods for toxic pyrrolizidine alkaloids by Colin Crews; Franz Berthiller; Rudi Krska (327-338).
Methods for the determination of toxic pyrrolizidine alkaloids in plants and foods are described with emphasis on the important aspects of sample extraction and clean-up and the now preferred determination by liquid chromatography–mass spectrometry. The efficiencies of different extraction solvents and methods are described, as are the methods of reduction of N-oxides. Appropriate liquid chromatography–mass spectrometry conditions are tabulated. This concise review is intended to guide analysts towards adopting a more unified and reliable approach to the analysis of these important toxins.
Keywords: Pyrrolizidine alkaloids; Review; LC–MS; SPE

Sample preparation is important for isolating desired components from complex matrices and greatly influences their reliable and accurate analysis. Recent trends in sample preparation include miniaturization, automation, high-throughput performance, online coupling with analytical instruments, and low-cost operation through extremely low or no solvent consumption. Microextraction techniques, such as liquid-phase microextraction and solid-phase microextraction, have these advantages over the traditional approaches of liquid–liquid extraction and conventional solid-phase extraction. This review focuses primarily on these microextraction techniques developed over the last decade, and presents a summary of the characteristics of various approaches in drug analysis.
Keywords: Sample preparation; Liquid-phase microextraction; Single-drop microextraction; Solid-phase microextraction; In-tube solid-phase microextraction; Drug analysis

Sources of contamination and remedial strategies in the multi-elemental trace analysis laboratory by Ilia Rodushkin; Emma Engström; Douglas C. Baxter (365-377).
In theory, state of the art inductively coupled plasma mass spectrometry (ICP–MS) instrumentation has the prerequisite sensitivity to carry out multi-elemental trace analyses at sub-ng L−1 to sub-pg L−1 levels in solution. In practice, constraints mainly imposed by various sources of contamination in the laboratory and the instrument itself, and the need to dilute sample solutions prior to analysis ultimately limit detection capabilities. Here we review these sources of contamination and, wherever possible, propose remedial strategies that we have found efficacious for ameliorating their impact on the results of multi-elemental trace analyses by ICP–MS. We conclude by providing a list of key points to consider when developing methods and preparing the laboratory to routinely meet the demands of multi-elemental analyses at trace analytical levels by ICP–MS.
Keywords: Inductively coupled plasma mass spectrometry; Trace analysis; Contamination prevention; Cleaning procedures

Biological matrices for the evaluation of exposure to environmental tobacco smoke during prenatal life and childhood by Heura Llaquet; Simona Pichini; Xavier Joya; Esther Papaseit; Oriol Vall; Julia Klein; Oscar Garcia-Algar (379-399).
The measurement of nicotine and its major metabolites cotinine and trans-3´-hydroxicotinine together with other minor metabolites (e.g., cotinine N-oxide, cotinine, and trans-3´-hydroxicotinine glucuronides) in conventional and nonconventional biological matrices has been used as a biomarker to assess the exposure to environmental tobacco smoke during childhood. The determination of these substances in matrices such as amniotic fluid, meconium, and fetal hair accounts for prenatal exposure to cigarette smoking at different stages of pregnancy. Nicotine and its metabolites in cord blood, neonatal urine, and breast milk are useful for determining acute exposure to drugs of abuse in the period immediately before and after delivery. Cotinine measurement in children’s blood and urine and nicotine and cotinine measurements in children’s hair constitute objective indexes of acute and chronic exposure during infancy, respectively. However, for monitoring and categorizing cumulative exposure to environmental tobacco smoke during the entire childhood, including the prenatal period, the assessment of nicotine in teeth has been proposed as a promising noninvasive tool. This article reviews the usefulness of measurement of nicotine and its metabolites in different fetal and pediatric biological matrices in light of noninvasive collection, time window of exposure detection, and finally clinical application in pediatrics.
Keywords: Biological matrices; Environmental tobacco smoke; Prenatal; Childhood

Dielectrophoresis for manipulation of micro/nano particles in microfluidic systems by C. Zhang; K. Khoshmanesh; A. Mitchell; K. Kalantar-zadeh (401-420).
Dielectrophoretic (DEP) force is exerted when a neutral particle is polarized in a non-uniform electric field, and depends on the dielectric properties of the particle and the suspending medium. The integration of DEP and microfluidic systems offers numerous applications for the separation, trapping, assembling, transportation, and characterization of micro/nano particles. This article reviews the applications of DEP forces in microfluidic systems. It presents the theory of dielectrophoresis, different configurations, and the applications of such systems for particle manipulation and device fabrication.
Keywords: Dielectrophoresis; Manipulation; Organic and inorganic particles; Microfluidics; Micro/nano scale

Two-dimensional liquid chromatography, 2D-LC of synthetic polymers is critically assessed. Similarities and differences of 2D-LC of low-molecular-mass and polymeric substances are reviewed. The rationale of application of 2D-LC to macromolecular substances is discussed. Basic information on retention mechanisms in liquid chromatography of synthetic polymers is furnished. The principles, reasons, and significance of coupling of retention mechanisms are explained. The resulting separation processes are elucidated, and the technical concepts of the corresponding experimental arrangements are described. The benefits of 2D-LC are demonstrated together with numerous problems and shortcomings of the method. Figure Schematic diagram of contour plot of the results of 2D-LC separation. A complex polymer system contains three constituents, each with both composition and molar mass distribution. Sample concentration is visualized by use of color, with concentration increasing from blue to yellow.
Keywords: Synthetic polymers; Liquid chromatography; Two-dimensional separations; Combinations of exclusion (entropic) and interaction (enthalpic) retention mechanisms

3-MCPD in food other than soy sauce or hydrolysed vegetable protein (HVP) by Ines Baer; Beatriz de la Calle; Philip Taylor (443-456).
This review gives an overview of current knowledge about 3-monochloropropane-1,2-diol (3-MCPD) formation and detection. Although 3-MCPD is often mentioned with regard to soy sauce and acid-hydrolysed vegetable protein (HVP), and much research has been done in that area, the emphasis here is placed on other foods. This contaminant can be found in a great variety of foodstuffs and is difficult to avoid in our daily nutrition. Despite its low concentration in most foods, its carcinogenic properties are of general concern. Its formation is a multivariate problem influenced by factors such as heat, moisture and sugar/lipid content, depending on the type of food and respective processing employed. Understanding the formation of this contaminant in food is fundamental to not only preventing or reducing it, but also developing efficient analytical methods of detecting it. Considering the differences between 3-MCPD-containing foods, and the need to test for the contaminant at different levels of food processing, one would expect a variety of analytical approaches. In this review, an attempt is made to provide an up-to-date list of available analytical methods and to highlight the differences among these techniques. Finally, the emergence of 3-MCPD esters and analytical techniques for them are also discussed here, although they are not the main focus of this review.
Keywords: 3-MCPD; Formation; Analytical determination; Foodstuff; Esters

An optimal design method for preventing air bubbles in high-temperature microfluidic devices by Tsuyoshi Nakayama; Ha Minh Hiep; Satoshi Furui; Yuji Yonezawa; Masato Saito; Yuzuru Takamura; Eiichi Tamiya (457-464).
DNA analysis with the polymerase chain reaction (PCR) has become a routine part of medical diagnostics, environmental inspections, food evaluations, and biological studies. Furthermore, the development of a microscale PCR chip is an essential component of studies aimed at integrating PCR into a micro total analysis system (μ-TAS). However, the occurrence of air bubbles in microchannels complicates this process. In this study, we investigated a new technique based on the fluid dynamics of laminar flow that utilizes a small amount of mineral oil at the beginning of sample injection to prevent air bubbles from occurring in microchannels. We also further optimized the pressure, the length of the pressurizing channel and the volume of oil, thus making our microfluidic device more useful for high-temperature PCR. Additionally, quantitative continuous-flow PCR was performed using the optimized PCR chip in order to detect genetically modified (GM) maize. DNA was extracted from GM maize, MON 810, and non-GM maize at several concentrations from 0% (w/v) to 100% (w/v). The DNA amplification signals were then analyzed on the PCR chip using a laser-based system. The signal from our microfluidic PCR chip was found to increase in direct proportion to the initial GM maize concentration.
Keywords: Microfluidic device; Continuous-flow PCR; Air bubbles; Genetically modified (GM) maize; Micro total analysis system (μ-TAS)

Sexing of turkey poults by Fourier transform infrared spectroscopy by Gerald Steiner; Thomas Bartels; Maria-Elisabeth Krautwald-Junghanns; Alois Boos; Edmund Koch (465-470).
Fourier transform infrared (FT-IR) spectroscopy was used to probe the molecular composition of germinal cells and to identify the gender of turkey poults. Germinal cells obtained from a feather pulp were characterized by FT-IR micro spectroscopy. The sample set consisted of growing contour feathers from 23 male and 23 female turkey poults. Significant spectral variations were observed in the range between 1,000 and 1,250 cm−1. The spectra of male turkey poults exhibit a significantly higher content of RNA than those of female turkeys. Spectral classification was performed by a non-supervised method based on the principal component analysis. An evaluation of the first and third PCs led to a classification of female and male poults with an accuracy of more than 95%.
Keywords: Bioanalytical methods; IR spectroscopy/Raman spectroscopy; Biological samples

Over the past years, ion mobility spectrometry (IMS) as a well established method within the fields of military and security has gained more and more interest for biological and medical applications. This highly sensitive and rapid separation technique was crucially enhanced by a multi-capillary column (MCC), pre-separation for complex samples. In order to unambiguously identify compounds in a complex sample, like breath, by IMS, a reference database is mandatory. To obtain a first set of reference data, 16 selected volatile organic substances were examined by MCC-IMS and comparatively analyzed by the standard technique for breath research, thermal desorption–gas chromatography–mass spectrometry. Experimentally determined MCC and GC retention times of these 16 compounds were aligned and their relation was expressed in a mathematical function. Using this function, a prognosis of the GC retention time can be given very precisely according to a recorded MCC retention time and vice versa. Thus, unknown MCC-IMS peaks from biological samples can be assigned—after alignment via the estimated GC retention time—to analytes identified by GC/MS from equivalent accomplished data. One example of applying the peak assignment strategy to a real breath sample is shown in detail.
Keywords: IMS; TD-GC/MS; Multi-capillary column (MCC); Volatile organic compound (VOC); Alignment; Breath analysis

Gene transcription analysis of carrot allergens by relative quantification with single and duplex reverse transcription real-time PCR by Jutta Zagon; Bärbel Jansen; Meike Knoppik; Anke Ehlers; Lothar W. Kroh; Thomas Holzhauser; Stefan Vieths; Hermann Broll (483-493).
Single and duplex real-time polymerase chain reaction (PCR) systems have been developed to quantify specific mRNA transcription of genes coding for the major Daucus carota allergen isoforms Dau c 1.01 and Dau c 1.02. Methods were tested with samples from the local market. Whereas the gene transcription levels for Dau c 1.01 were consistently high in all investigated samples, significant differences for the Dau c 1.02 transcription could be demonstrated in randomly collected market samples. The gene transcription level for the minor Dau c 1.02 variant is about one log below Dau c 1.01. Both formats, single or duplex real-time methods, exhibit ideal cycle threshold (CT) ranges and good reproducibility. In particular, the easily performed duplex real-time PCR system is potentially suited for the selection of hypoallergenic varieties and studying the impact of post-harvesting or environmental conditions.
Keywords: Reverse transcriptase; Real-time PCR; Daucus carota ; Allergens; Dau c 1.01; Dau c 1.02; PCR; Gene expression

This article describes the use of microfluidic paper-based analytical devices (μPADs) to perform quantitative chemical assays with internal standards. MicroPADs are well-suited for colorimetric biochemical assays; however, errors can be introduced from the background color of the paper due to batch difference and age, and from color measurement devices. To reduce errors from these sources, a series of standard analyte solutions and the sample solution are assayed on a single device with multiple detection zones simultaneously; an analyte concentration calibration curve can thus be established from the standards. Since the μPAD design allows the colorimetric measurements of the standards and the sample to be conducted simultaneously and under the same condition, errors from the above sources can be minimized. The analytical approach reported in this work shows that μPADs can perform quantitative chemical analysis at very low cost. Figure The scanned images of microfluidic paper-based analytical devices for colorimetric multi-analyte detection (left) and quantitative biomarker assay (right)
Keywords: Quantitative assay; Paper; Microfluidic devices; Biomarkers

Capabilities of laboratories to determine melamine in food—results of an international proficiency test by Andreas Breidbach; Katrien Bouten; Katy Kröger; Franz Ulberth (503-510).
A proficiency test to assess the capabilities of laboratories to determine melamine in a milk powder and a baking mix, representing starch-containing foods like bread and biscuits, was carried out in January 2009. The need for such an interlaboratory comparison arose from a health scare in China about melamine-tainted powdered milk in the second half of 2008. Laboratories in 31 countries, including Australia, China, India, Japan, New Zealand and the USA, and 21 of the 27 Member States of the European Union participated and reported back 114 results for the milk powder and 112 for the baking mix test materials. The reported results were compared to reference values determined by exact-matching double isotope dilution mass spectrometry. The so-determined assigned values were 10.0 ± 0.6 mg/kg melamine in the milk powder and 3.18 ± 0.17 mg/kg melamine in the baking mix. A coverage factor k of 2 was applied to calculate the expanded uncertainties. Three quarters of all reported results for both materials had associated z scores which were satisfactory (z ≤ |2|). Of the reported results, 90% was accompanied by a measurement uncertainty statement, and the majority of the measurement uncertainty ranges were reasonable. A number of laboratories were found to underestimate their measurement uncertainties. Methods that involved the use of stable-isotope-labelled melamine were shown to be clearly advantageous with regard to the accuracy of the results. However, no significant influence by other method parameters could be identified.
Keywords: Melamine; Proficiency test; Isotope dilution mass spectrometry

Bonded ionic liquid polymeric material for solid-phase microextraction GC analysis by Eranda Wanigasekara; Sirantha Perera; Jeffrey A. Crank; Leonard Sidisky; Robert Shirey; Alain Berthod; Daniel W. Armstrong (511-524).
Four new ionic liquids (IL) were prepared and bonded onto 5-µm silica particles for use as adsorbent in solid-phase microextraction (SPME). Two ILs contained styrene units that allowed for polymerization and higher carbon content of the bonded silica particles. Two polymeric ILs differing by their anion were used to prepare two SPME fibers that were used in both headspace and immersion extractions and compared to commercial fibers. In both sets of experiments, ethyl acetate was used as an internal standard to take into account adsorbent volume differences between the fibers. The polymeric IL fibers are very efficient in headspace SPME for short-chain alcohols. Immersion SPME also can be used with the IL fibers for short-chain alcohols as well as for polar and basic amines that can be extracted at pH 11 without damage to the IL-bonded silica particles. The sensitivities of the two IL fibers differing by the anion were similar. Their efficacy compares favorably to that of commercial fibers for polar analytes. The mechanical strength and durability of the polymeric IL fibers were excellent. Figure Chemistry of the polymerized ionic liquid absorbant and its morphology when bonded to the SPME fiber.
Keywords: Solid-phase microextraction; Headspace; Immersion; Ionic liquid; Bonded phase; Beverages; Gas chromatography

Near-infrared (NIR) Raman spectroscopy was used to measure spectra of dried human blood samples from multiple donors. Two major questions addressed in this paper involve the influence of sample heterogeneity and potential Raman spectral variations that could arise between different donors of blood. Advanced statistical analysis of spectra obtained from multiple spots on dry samples showed that dry blood is chemically heterogeneous, and its Raman spectra could be presented very well as a linear combination of a fluorescent background and two Raman spectroscopic components that are dominated by hemoglobin and fibrin, respectively. Each sample Raman spectrum contains the same major peaks, but the relative contribution of the hemoglobin and fibrin components varies with the donor. Therefore, no single spectrum could adequately represent an experimental Raman spectrum of dry blood in a quantitative way, but rather the combination of hemoglobin and fibrin spectral components could be considered to be a spectroscopic signature for blood. This proof-of-concept approach shows the potential for Raman spectroscopy to be used in forensic analysis to identify an unknown substance such as blood.
Keywords: Raman spectroscopy; Blood; Principal components; Statistical analysis; Forensic science; Body fluid identification

This paper describes a simple new approach toward improving resolution of two-dimensional (2-D) protein gels used to explore the mammalian proteome. The method employs sample prefractionation using solution-phase isoelectric focusing (IEF) to split the mammalian proteome into well-resolved pools. As crude samples are thus prefractionated by pI range, very-narrow-pH-range 2-D gels can be subsequently employed for protein separation. Using custom pH partition membranes and commercially available immobilized pH gradient (IPG) strips, we maximized the total separation distance and throughput of seven samples obtained by prefractionation. Both protein loading capacity and separation quality were higher than the values obtained by separation of fractionated samples on narrow-pH-range 2-D gels; the total effective IEF separation distance was ~82 cm over the pH range pH 3–10. This improved method for analyzing prefractionated samples on narrow-pH-range 2-D gels allows high protein resolution without the use of large gels, resulting in decreased costs and run times. Figure Optimizing 2-D gel size, resolution, and throughput for analysis of 0.5 pH-unit ZOOM™ IEF fractions. Aliquots of 0.5-pHunit fractions (pH 4.9–5.4) from a prefractionated sample of human breast cancer MCF-7/6 cell extract were separated on commercial 24-cm (a) or 18-cm (b) (pH 4.5-5.5) gels. After IEF, second-dimension gels of various sizes were evaluated. IPG strips could be applied directly to larger-sized second-dimension gels, or excess IPG gel regions (encompassing pH values outside the fractionated pH range) could be trimmed so that the IPG gels (now covering ~0.7 pH units) could be applied to smaller sized second-dimension gels. All seconddimension separations were performed using 10% (w/v) Tris–tricine SDS gels, followed by silver staining. Protein loading was adjusted with respect to gel volume to obtain similar staining intensities. Fractionated samples equivalent to the following amounts of original cell extract were used (from left to right): 150μg, 100μg, 100μg, and 25μg
Keywords: High-throughput; Sample prefractionation method; Narrow-pH-range 2-D gel; Separation and resolution