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Analytical and Bioanalytical Chemistry (v.387, #1)
Single cell analysis by Jonathan V. Sweedler; Edgar A. Arriaga (pp. 1-2).
is a Lycan Professor of Chemistry at the University of Illinois (USA) and is associated with the Institute of Genomic Biology, the Beckman Institute, Biotechnology Center, the Neuroscience Program, and the Bioengineering Program at the UI. His research interests are in bioanalytical chemistry, and focus on developing new methods for assaying nanoliter volume samples such as single cells, and applying these methods to study the distribution and dynamic release of neurotransmitters and neuropeptides from individual neurons. Using single-cell capillary electrophoresis and mass spectrometry, he is investigating the roles that peptide hormones and neurotransmitters play in behavior, learning and memory. is an Associate Professor of Chemistry at the University of Minnesota (USA) and graduate faculty in the Department of Biomedical Engineering (UMN). His interests include the development of analytical methods for investigating subcellular properties based on chemical imaging, microseparations, and mass spectrometry. Using capillary electrophoresis to measure properties of isolated individual organelles, his research group is investigating the subcellular distribution of xenobiotics and the role of organelles in biological aging.
Choosing one from the many: selection and sorting strategies for single adherent cells by Christopher E. Sims; Mark Bachman; G. P. Li; Nancy L. Allbritton (pp. 5-8).
Single-cell manipulation and analysis using microfluidic devices
by Gregory T. Roman; Yanli Chen; Pernilla Viberg; Anne H. Culbertson; Christopher T. Culbertson (pp. 9-12).
Multiple sampling in single-cell enzyme assays using capillary electrophoresis with laser-induced fluorescence detection
by Glen K. Shoemaker; Monica M. Palcic (pp. 13-15).
Advances in immunoanalysis of single cells with capillary electrophoresis
by Hua Zhang; Shuge Tian (pp. 21-23).
Mass spectrometric charting of neuropeptides in arthropod neurons
by Stephanie S. DeKeyser; Lingjun Li (pp. 29-35).
Spectrally resolved fluorescence lifetime imaging microscopy (SFLIM)—an appropriate method for imaging single molecules in living cells
by Jens-Peter Knemeyer; Nicole Marmé; Jörg D. Hoheisel (pp. 37-40).
Single-molecule tracking in eukaryotic cell nuclei
by Jan Peter Siebrasse; David Grünwald; Ulrich Kubitscheck (pp. 41-44).
Recent advances in capillary electrophoretic analysis of individual cells by Imee G. Arcibal; Michael F. Santillo; Andrew G. Ewing (pp. 51-57).
Because variability exists within populations of cells, single-cell analysis has become increasingly important for probing complex cellular environments. Capillary electrophoresis (CE) is an excellent technique for identifying and quantifying the contents of single cells owing to its small volume requirements and fast, efficient separations with highly sensitive detection. Recent progress in both whole-cell and subcellular sampling has allowed researchers to study cellular function in the areas of neuroscience, oncology, enzymology, immunology, and gene expression.
Keywords: Single-cell analysis; Cell sampling; Capillary electrophoresis
mRNA transport to and translation in neuronal dendrites by Chia-wen K. Wu; Fanyi Zeng; James Eberwine (pp. 59-62).
Transport of mRNA is an important biological process in all cells that sets up gradients of translated protein from the site of mRNA docking and translation. Neurons are highly polarized cells where the targeted movement of RNAs and local translation at that site have been shown to be integral to the proper functioning of the neuron. Indeed, this specialized biological function for localized RNAs in particular neurons may in part confer a selective advantage on these cells such that they “out-compete” others in the race to establish synaptic connectivity. In this mini-review we highlight some of the salient features of RNA targeting and translation in neurons.
Keywords: Nucleic acids (DNA/RNA); Cell systems/single-cell analysis; Biological samples
Real-time dynamics of label-free single mast cell granules revealed by differential interference contrast microscopy by Hung-Wing Li; Michael McCloskey; Yan He; Edward S. Yeung (pp. 63-69).
We demonstrate the capability of differential interference contrast (DIC) microscopy as a simple and useful tool for studying cellular events without fluorescence labeling. By coupling an advanced DIC microscope to a computer-controlled motorized vertical stage and a high-speed, high-resolution CCD camera, real-time three-dimensional monitoring is possible in a high-throughput manner. The performance among three modes of microscopy, bright-field, dark-field and DIC, in terms of horizontal resolving power and vertical sectioning was investigated. As a model, exocytosis of rat peritoneal mast cells was recorded on the subsecond time scale. Three-dimensional tracking of granules during degranulation was achieved and granule–granule fusion before plasma membrane fusion was recorded.
Keywords: Microscopy; Imaging; Mast cells; CCD camera
Monitoring dynamic systems with multiparameter fluorescence imaging by Volodymyr Kudryavtsev; Suren Felekyan; Anna K. Woźniak; Marcelle König; Carl Sandhagen; Ralf Kühnemuth; Claus A. M. Seidel; Filipp Oesterhelt (pp. 71-82).
A new general strategy based on the use of multiparameter fluorescence detection (MFD) to register and quantitatively analyse fluorescence images is introduced. Multiparameter fluorescence imaging (MFDi) uses pulsed excitation, time-correlated single-photon counting and a special pixel clock to simultaneously monitor the changes in the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time. The three spatial coordinates are also stored. The most statistically efficient techniques known from single-molecule spectroscopy are used to estimate fluorescence parameters of interest for all pixels, not just for the regions of interest. Their statistical significance is judged from a stack of two-dimensional histograms. In this way, specific pixels can be selected for subsequent pixel-based subensemble analysis in order to improve the statistical accuracy of the parameters estimated. MFDi avoids the need for sequential measurements, because the registered data allow one to perform many analysis techniques, such as fluorescence-intensity distribution analysis (FIDA) and fluorescence correlation spectroscopy (FCS), in an off-line mode. The limitations of FCS for counting molecules and monitoring dynamics are discussed. To demonstrate the ability of our technique, we analysed two systems: (i) interactions of the fluorescent dye Rhodamine 110 inside and outside of a glutathione sepharose bead, and (ii) microtubule dynamics in live yeast cells of Schizosaccharomyces pombe using a fusion protein of Green Fluorescent Protein (GFP) with Minichromosome Altered Loss Protein 3 (Mal3), which is involved in the dynamic cycle of polymerising and depolymerising microtubules.
Keywords: Multiparameter fluorescence imaging; Fluorescence correlation spectroscopy; FCS; Confocal microscopy; Lifetime imaging; Microtubules
Analysis of subcellular surface structure, function and dynamics by D. Anselmetti; N. Hansmeier; J. Kalinowski; J. Martini; T. Merkle; R. Palmisano; R. Ros; K. Schmied; A. Sischka; K. Toensing (pp. 83-89).
Analytics of single biological cells allows quantitative investigation from a structural, functional and dynamical point of view and opens novel possibilities to an unamplified subcellular analysis. In this article, we report on three different experimental methods and their applications to single cellular systems with a subcellular sensitivity down to the single molecule level. First, the subcellular surface structure of living bacteria (Corynebacterium glutamicum) was investigated with atomic force microscopy (AFM) at the resolution of individual surface layer (S-layer) proteins; discrimination of bacterial strains that lack the expression of hexagonally packed surface layer proteins was possible. Second, quantitative measurement of individual recognition events of membrane-bound receptors on living B-cells was achieved in single cell manipulation and probing experiments with optical tweezers (OT) force spectroscopy. And third, intracellular dynamics of translocating photoactivatable GFP in plant protoplasts (Nicotiana tabacum BY-2) was quantitatively monitored by two-photon laser scanning microscopy (2PLSM).
Keywords: Atomic force microscopy; Optical tweezers; Two-photon laser scanning microscopy
Cell lysis inside the capillary facilitated by transverse diffusion of laminar flow profiles (TDLFP) by Maxim V. Berezovski; Tak W. Mak; Sergey N. Krylov (pp. 91-96).
Chemical cytometry studies the molecular composition of individual cells by means of capillary electrophoresis or capillary chromatography. In one of its realizations an intact cell is injected inside the capillary, the plasma membrane is disrupted to release the cellular contents into the separation buffer, and, finally, the molecules of interest are separated and detected. The solubilization of the plasma membrane with a surfactant is a simple and efficient way of achieving cell lysis inside the capillary. To facilitate cell lysis by a surfactant the cell has to be contacted with the surfactant inside the capillary. We recently introduced a generic method for mixing solutions inside the capillary termed transverse diffusion of laminar flow profiles (TDLFP). In this work, we propose that TDLFP can facilitate efficient cell lysis inside the capillary. Conceptually, a short plug of the surfactant is injected by pressure prior to cell injection. The cell is then injected by pressure wizthin a plug of the physiological buffer. Due to the parabolic profiles of pressure-driven laminar flows the interface between the plug of the surfactant and that of the physiological buffer is predominantly longitudinal. Transverse diffusion mixes the surfactant with the physiological buffer, which leads to surfactant’s contact with the cell and subsequent cell lysis. Here, we demonstrate that the proposed concept is valid. TDLFP-facilitated cell lysis by a short plug of the surfactant allows us to exclude the surfactant from the run buffer, and, hence, facilitates modes of separation, which are incompatible with the surfactant’s presence in the run buffer. In addition to cell lysis, TDLFP will be used to mix the cellular components with labeling reactants, affinity probes, inhibitors, etc. We foresee that the generic nature and enabling capabilities of TDLFP will speed up the maturation of chemical cytometry into a practical bioanalytical tool.
Keywords: Single-cell analysis; Chemical cytometry; Cell lysis; Transverse diffusion of laminar flow profiles
A multichannel native fluorescence detection system for capillary electrophoretic analysis of neurotransmitters in single neurons by T. Lapainis; C. Scanlan; S. S. Rubakhin; J. V. Sweedler (pp. 97-105).
A laser-induced native fluorescence detection system optimized for analysis of indolamines and catecholamines by capillary electrophoresis is described. A hollow-cathode metal vapor laser emitting at 224 nm is used for fluorescence excitation, and the emitted fluorescence is spectrally distributed by a series of dichroic beam-splitters into three wavelength channels: 250–310 nm, 310–400 nm, and >400 nm. A separate photomultiplier tube is used for detection of the fluorescence in each of the three wavelength ranges. The instrument provides more information than a single-channel system, without the complexity associfated with a spectrograph/charge-coupled device-based detector. With this instrument, analytes can be separated and identified not only on the basis of their electrophoretic migration time but also on the basis of their multichannel signature, which consists of the ratios of relative fluorescence intensities detected in each wavelength channel. The 224-nm excitation channel resulted in a detection limit of 40 nmol L−1 for dopamine. The utility of this instrument for single-cell analysis was demonstrated by the detection and identification of the neurotransmitters in serotonergic LPeD1 and dopaminergic RPeD1 neurons, isolated from the central nervous system of the well-established neurobiological model Lymnaea stagnalis. Not only can this system detect neurotransmitters in these individual neurons with S/N>50, but analyte identity is confirmed on the basis of spectral characteristics.
Keywords: Capillary electrophoresis; Laser-induced native fluorescence detection; Single-cell analysis; Dopamine; Lymnaea stagnalis
Analysis of mitochondria isolated from single cells by Ryan D. Johnson; Marian Navratil; Bobby G. Poe; Guohua Xiong; Karen J. Olson; Hossein Ahmadzadeh; Dmitry Andreyev; Ciarán F. Duffy; Edgar A. Arriaga (pp. 107-118).
Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrion-specific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.
Keywords: Capillary electrophoresis; Bioanalytical methods; Cell systems; Single-cell analysis; Fluorescence; Luminescence; Bioassays
Analysis of oxidized multi-walled carbon nanotubes in single K562 cells by capillary electrophoresis with laser-induced fluorescence by Hua Xiao; Lisong Yang; Hanfa Zou; Ling Yang; X. Chris Le (pp. 119-126).
Short oxidized multi-walled carbon nanotubes (CNT) were derivatized with fluorescein isothiocyanate (FITC). Capillary electrophoresis coupled with laser-induced fluorescence (CE–LIF) was then used to separate and detect the fluorescently labeled carbon-nanotube probes (CNTP) in multidrug-resistant cells (K562A) and the parent cells (K562S). Greater expression of P-glycoprotein in K562A cells than in K562S cells was confirmed by use of anti-P-glycoprotein antibody and flow-cytometric analysis. Analyses of CNTP in both cell lines using both CE–LIF and flow cytometry showed that CNTP could traverse the cellular membrane without being pumped out by P-glycoprotein. The CNTP distributed in both cell lines was analyzed at the single cell level and the results were compared with those from analysis of ten cells and of the lysate from bulk cells. The results revealed the CE–LIF method could be used for quantitative analysis of CNT in single cells in studies of drug delivery and multidrug resistance.
Keywords: Multi-walled carbon nanotubes; Single-cell analysis; Multidrug resistance; Capillary electrophoresis; Laser-induced fluorescence
Health care: another challenge for analytical chemistry by Günter Gauglitz (pp. 127-128).
is Professor at the Eberhard-Karls University of Tübingen, working in analytical and physical chemistry. He is chairman of the GDCh Division of Analytical Chemistry. For the last ten years, his main scientific interests have centered on research and development in the area of chemical and biochemical sensors with special focus on the characterization of interfaces of polymers and biomembranes, application of surface spectroscopic techniques and spectral interferometry to monitor changes in optical thickness of thin layers, and effects of Fresnel reflectivity at interfaces
Publication of chemical research: do we need ethical standards? by Bo Karlberg (pp. 129-130).
is Professor of Analytical Chemistry at Stockholm University, Sweden. He is also the Chairman of the Division of Analytical Chemistry (DAC) within the European Association for Chemical and Molecular Sciences (EuCheMS). His research interest lies in the areas of vibrational spectroscopy, flow injection analysis and capillary electrophoresis.
Diversity and comparability—towards a common European Higher Education Area by Martin Vogel (pp. 131-133).
is a member of the Department of Analytical Chemistry at the University of Münster. His main research interests are the development of methods for forensic analysis, the investigation of ionisation mechanisms in LC/MS, and the screening of enzymatic activities in complex samples.
Reporting results of biomonitoring studies by Warren G. Foster; John Agzarian (pp. 137-140).
is the CIHR/Ontario Women’s Health Council Professor and Director of the Centre for Reproductive Care & Reproductive Biology Division in the Department of Obstetrics & Gynecology at McMaster University. His research program is focused on determining the cellular and molecular mechanisms of environmental toxicant effects on estrogen-sensitive target tissues. He has conducted numerous studies to characterize human exposure to environmental toxicants, results of which are used to guide dose selection in animal and tissue culture studies.
Towards automated, miniaturized and solvent-free sample preparation methods
by Frank David; Els Van Hoeck; Pat Sandra (pp. 141-144).
Mycotoxin analysis: state-of-the-art and future trends
by Rudolf Krska; Alexandra Molinelli (pp. 145-148).
Femtosecond laser ablation inductively coupled plasma mass spectrometry: achievements and remaining problems
by J. Koch; D. Günther (pp. 149-153).
Temperature gradient focusing for microchannel separations
by Jonathan G. Shackman; Matthew S. Munson; David Ross (pp. 155-158).
Advantages of downsizing electrochemical detection for DNA assays
by Ingrid Fritsch; Zoraida P. Aguilar (pp. 159-163).
Trends in the analytical applications of chemiluminescence in the liquid phase
by Ana M. García-Campaña; Francisco J. Lara (pp. 165-169).
Methods developed for SELEX by Subash Chandra Bose Gopinath (pp. 171-182).
SELEX (systematic evolution of ligands by exponential enrichment) is a process that involves the progressive purification from a combinatorial library of nucleic acid ligands with a high affinity for a particular target by repeated rounds of partitioning and amplification. With the development of aptamer technology over the last decade, various modified SELEX processes have arisen that allow various aptamers to be developed against a wide variety of molecules, irrespective of the target size. In the present review, the separation methods used in such SELEX processes are reviewed.
Keywords: Aptamer; DNA; RNA; SELEX; Separation
Recent developments in optical detection methods for microchip separations by Sebastian Götz; Uwe Karst (pp. 183-192).
This paper summarizes the features and performances of optical detection systems currently applied in order to monitor separations on microchip devices. Fluorescence detection, which delivers very high sensitivity and selectivity, is still the most widely applied method of detection. Instruments utilizing laser-induced fluorescence (LIF) and lamp-based fluorescence along with recent applications of light-emitting diodes (LED) as excitation sources are also covered in this paper. Since chemiluminescence detection can be achieved using extremely simple devices which no longer require light sources and optical components for focusing and collimation, interesting approaches based on this technique are presented, too. Although UV/vis absorbance is a detection method that is commonly used in standard desktop electrophoresis and liquid chromatography instruments, it has not yet reached the same level of popularity for microchip applications. Current applications of UV/vis absorbance detection to microchip separations and innovative approaches that increase sensitivity are described. This article, which contains 85 references, focuses on developments and applications published within the last three years, points out exciting new approaches, and provides future perspectives on this field.
Keywords: Optical detection methods; Microchip; Microfluidic; Separation; Review
Recent developments in methods and technology for analysis of biological samples by MALDI-TOF-MS by Chensong Pan; Songyun Xu; Houjiang Zhou; Yu Fu; Mingliang Ye; Hanfa Zou (pp. 193-204).
Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF-MS) is widely used in a variety of fields because it has the characteristics of speed, ease of use, high sensitivity, and wide detectable mass range for obtaining molecular weights and for structural characterization of macromolecules. In this article we summarize recent developments in matrix additives, new matrices, and sample-pretreatment methods using off-probe or on-probe techniques or nanomaterials for MALDI-TOF-MS analysis of biological samples.
Keywords: Review; MALDI; Sample pretreatment; Matrix; Nanomaterials
Optical immunosensors for environmental monitoring: How far have we come? by M. A. González-Martínez; R. Puchades; A. Maquieira (pp. 205-218).
Immunosensing has proved to be a very interesting research area. This review discusses what has actually been achieved in the field of optical immunosensing for environmental screening, and what still needs to be done. The review is presented from a practical point of view. In terms of the basic design of the immunosensor, there is a trend towards decreasing assay time; indeed, this has been reduced from 15–20 minutes to less than 5 minutes. Another goal is to simplify the manifold, and label-free approaches combining indirect assay formats and the detection of antibody binding are popular. Rapid displacement assays have also been investigated thoroughly. In terms of some important features of immunosensing devices, the reusability of the sensing element has been studied in great depth, and working lifetimes of more than five hundred assays can now be found for all assay formats. Multianalyte assays are now being investigated, and current systems are able to monitor 2–3 target compounds, although this number is set to increase greatly (to >30) in the near future. In this sense, an increasing number of publications can be found on microarrays intended for multianalyte determinations. The application of immunosensing to real situations is the main challenge. Immunosensors are barely commercialized and are yet to be established as research or routine tools, due to a lack of validated protocols for a wide range of sample matrices. Regarding compounds considered as analytes, some significant pollutants such as dioxins or pharmaceuticals are rarely chosen as targets, although the current tendency is towards a broader spectrum of analytes. New immunoreagents should be raised for these compounds, for use in immunosensors that can be used as screening tools.
Keywords: Immunosensor; Optical; Environment
Determination of airborne isocyanates by Hartmut Henneken; Martin Vogel; Uwe Karst (pp. 219-236).
Isocyanates are important in industrial hygiene and workplace monitoring. Owing to their severe acute toxicity and sensitizing properties, analytical methods with high sampling efficiency and sensitivity in the low ppb to ppt range are required. The reactivity of isocyanates necessitates initial derivatization with nucleophilic agents—usually amines—for stabilization and enrichment; this is often followed by chromatographic separation with spectroscopic, electrochemical, or mass spectrometric detection. Sampling strategies for airborne isocyanates comprise active, i.e. pumped, or passive, i.e. diffusive, methods; the method selected depends on the application. Whereas active methods rely mainly on impingers, reagent-coated filters, or sampling tubes, passive samplers make use of reagent-coated filters, the surface of which is connected to the air sample by diffusion channels. Because airborne isocyanates are prone to occur in different forms, i.e. as vapors, as aerosols, or adsorbed on particulate matter, denuder sampling has been introduced, thus enabling simultaneous collection of gaseous and aerosol isocyanates. The first part of this review summarizes chemical methods and reagents which have been introduced for derivatization of airborne isocyanates. The advantages and drawbacks of the individual derivatization procedures and their combination with different detection principles are evaluated. In the second part, the most recent developments in air sampling for isocyanates, with special focus on diffusive sampling, are reviewed and critically discussed.
Keywords: Isocyanates; Air analysis; Sampling; Derivatization; Liquid chromatography
Sensor technology and its application in environmental analysis by Peter A. Lieberzeit; Franz L. Dickert (pp. 237-247).
Environmental analysis is one of the fundamental applications of chemical sensors. In this review we describe different sensor systems for the gas and liquid phases that have been tested either with real-life samples or in the field during the last five years. Most field sensors rely either on electrochemical or optical transducers. In the gas phase, systems have been proposed for analysis of oxides of nitrogen, carbon, and sulfur in air, and volatile organic compounds. In the liquid phase, most detection systems used for real-life samples detect heavy-metal ions or organic contamination, for example pesticides, organic solvents and polycyclic aromatic hydrocarbons. Figure Chemical sensors for real-life environmental applications
Keywords: Chemical sensors; Environmental analysis; Field measurements; Gas sensing; Liquid sensing
Ultrasound in analytical chemistry by F. Priego Capote; M. D. Luque de Castro (pp. 249-257).
Ultrasound is a type of energy which can help analytical chemists in almost all their laboratory tasks, from cleaning to detection. A generic view of the different steps which can be assisted by ultrasound is given here. These steps include preliminary operations usually not considered in most analytical methods (e.g. cleaning, degassing, and atomization), sample preparation being the main area of application. In sample preparation ultrasound is used to assist solid-sample treatment (e.g. digestion, leaching, slurry formation) and liquid-sample preparation (e.g. liquid–liquid extraction, emulsification, homogenization) or to promote heterogeneous sample treatment (e.g. filtration, aggregation, dissolution of solids, crystallization, precipitation, defoaming, degassing). Detection techniques based on use of ultrasonic radiation, the principles on which they are based, responses, and the quantities measured are also discussed.
Keywords: Ultrasound; Analytical process; Sample preparation; Ultrasound-based detection techniques
Microfabrication of screen-printed nanoliter vials with embedded surface-modified electrodes by Jeffrey S. Lenihan; J. Christopher Ball; Vasilis G. Gavalas; Janet K. Lumpp; John Hines; Sylvia Daunert; Leonidas G. Bachas (pp. 259-265).
A self-contained ion-selective sensing system within a nanoliter-volume vial has been developed by integrating screen printing, laser ablation, and molecular imprinting techniques. Screen printing and laser ablation are used in tandem to fabricate nanoliter-volume vials with carbon and Ag/AgCl ring electrodes embedded in the sidewalls. Using multisweep cyclic voltammetry, the surface of the carbon electrode can be modified with a polypyrrole film. By polymerizing pyrrole in the presence of nitrate, pores complementary to the nitrate anion in size, shape, and charge distribution are formed in the resulting film. Electrochemical cells modified with this nitrate-imprinted polypyrrole film show a near-Nernstian response to nitrate, and excellent reproducibility. The integration of molecular recognition and electrochemical response in the nanoliter vials is demonstrated by the detection of as little as 0.36 ng nitrate in nanoliter-volume samples. The integration of tailored molecular recognition within nanoliter vials via established fabrication and imprinting protocols should result in a number of nanosensor devices with applications in BioMEMS and micro total analysis systems.
Keywords: Microfabrication; Nanoliter vials; Ion-selective sensing
Disposable microfluidic ELISA for the rapid determination of folic acid content in food products by Daniela Hoegger; Patrick Morier; Christine Vollet; Dominique Heini; Frédéric Reymond; Joël S. Rossier (pp. 267-275).
A micro-analytical system for rapid and quantitative analysis by inhibition immunoassay is presented and applied to the detection of folic acid. Eight polymer microchannels of 65-nL volume each and containing microelectrodes are embedded in a cartridge so that they can be operated simultaneously. All fluidic steps as well as the amperometric detection in the channels are operated by an instrument and software developed in-house. The fluidic steps of the immunoassay occur through hydrodynamic loading of the different solutions through the channels. The speed and duration of the flow and incubation parameters can thus be adapted to the biological and testing requirements. The effectiveness of the system was demonstrated by analysing folic acid concentrations in real infant formula samples within 5 min. In an effort to get a fully monitored assay, each fluidic step is monitored thanks to continuous amperometric detection of oxygen in the microchannel. ELISA performed in polymer microfluidic channels with electrochemical detection
Keywords: Electroanalytical methods; Bioassays; Immunoassays; ELISA; Biosensors; Enzymes; Microfluidics; Microfabrication
A microfluidic system for evaluation of antioxidant capacity based on a peroxyoxalate chemiluminescence assay by Maliwan Amatatongchai; Oliver Hofmann; Duangjai Nacapricha; Orawon Chailapakul; Andrew J. deMello (pp. 277-285).
A microfluidic system incorporating chemiluminescence detection is reported as a new tool for measuring antioxidant capacity. The detection is based on a peroxyoxalate chemiluminescence (PO-CL) assay with 9,10-bis-(phenylethynyl)anthracene (BPEA) as the fluorescent probe and hydrogen peroxide as the oxidant. Antioxidant plugs injected into the hydrogen peroxide stream result in inhibition of the CL emission which can be quantified and correlated with antioxidant capacity. The PO-CL assay is performed in 800-μm-wide and 800-μm-deep microchannels on a poly(dimethylsiloxane) (PDMS) microchip. Controlled injection of the antioxidant plugs is performed through an injection valve. Of the plant-food based antioxidants tested, β-carotene was found to be the most efficient hydrogen peroxide scavenger (SA HP of 3.27 × 10−3 μmol−1 L), followed by α-tocopherol (SA HP of 2.36 × 10−3 μmol−1 L) and quercetin (SA HP of 0.31 × 10−3 μmol−1 L). Although the method is inherently simple and rapid, excellent analytical performance is afforded in terms of sensitivity, dynamic range, and precision, with RSD values typically below 1.5%. We expect our microfluidic devices to be used for in-the-field antioxidant capacity screening of plant-sourced food and pharmaceutical supplements. Figure Assembled PDMS microchip sandwiched between two glass plates with the top plate containing capillary reservoirs
Keywords: Antioxidant capacity; Peroxyoxalate chemiluminescence; Microfluidics
On-line monitoring of pH junctions in capillary electrophoresis using Fourier transform infrared spectrometry by Guillermo Quintás; Elena Nuñez; M. Vellekoop; Bernhard Lendl (pp. 287-292).
Fourier transform mid-infrared spectroscopic detection is proposed as an on-line detection technique for the study of on-line preconcentration processes in capillary electrophoresis (CE). The molecule-specific information contained in mid-IR spectra can be used to directly determine the chemical compositions of individual zones and their boundaries. This paper reports on pH junctions employed in myoglobin analysis. On-line mid-IR detection allowed the shape of the sample peak to be monitored as well as the chemical compositions of the surrounding zones. From this information it was possible to obtain detailed insights into the actual chemical compositions of the individual zones governing the efficiency of the preconcentration technique applied. The principle of measurement outlined here can therefore also be regarded as a promising one for investigating other on-line preconcentration techniques, like stacking, sweeping, and pH junction-sweeping among others. Fourier transform mid-infrared spectroscopic detection has been employed in pH junction experiments. This approach can be used to measure the chemical compositions of the phase boundaries formed, as well as the relative positions of the analyte in the zones. The principle of this technique is demonstrated by measuring myoglobin (acetate buffer, pH 4.5) in an ammonium BGE (pH 9.25)
Keywords: CE; FTIR; Myoglobin; pH junction
Direct sample injection for capillary electrophoretic determination of organic acids in cerebrospinal fluid by Rawi Ramautar; Govert W. Somsen; Gerhardus J. de Jong (pp. 293-301).
Organic acids in cerebrospinal fluid (CSF) are potential diagnostic markers for neurological diseases and metabolic disorders. A capillary electrophoretic (CE) method for the direct analysis, i.e., without any sample preparation, of six organic acids in CSF was developed. A capillary coating consisting of a triple layer of charged polymers (polybrene-dextran sulfate-polybrene) was used in combination with a negative separation voltage, providing fast and efficient analysis of acidic compounds. Separation conditions, such as background electrolyte (BGE) concentration and pH were optimized, and the influence of albumin and sodium chloride was systematically studied using a set of test compounds. With injection volumes of ca. 44 nL, plate numbers of up to ca. 150,000 were obtained with a BGE of 200 mM sodium phosphate (pH 6.0). It appeared that high sodium chloride concentrations in the sample hardly affected the peak width and shape of the organic acids, most probably due to transient isotachophoresis effects occurring in the sample zone. Adverse effects of CSF proteins, which frequently compromise the CE performance, could be effectively minimized by the triple layer coating in combination with rinses of 0.1 M hydrochloric acid. Overall, the developed CE system allowed direct injections of CSF samples, yielding good separation efficiencies and stable migration times (RSDs < 2%) for organic acids. Validation of the method with artificial and real CSF samples showed good linear responses (r > 0.99), and LODs for the organic acids were in the range of 2–8 μg/mL when applying UV detection. RSDs for migration times and peak areas were <2% and <7%, respectively. The applicability of the CE system is shown for the determination of organic acids in CSF samples.
Keywords: Capillary electrophoresis; Triple layer coating; Organic acids; Cerebrospinal fluid; Direct injection
Electron-transfer properties of different carbon nanotube materials, and their use in glucose biosensors by Yanli Yao; Kwok-Keung Shiu (pp. 303-309).
Different types of carbon nanotube material (single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) of different internal diameter) have been used for preparation of CNT-modified glassy-carbon electrodes. Redox reactions involving ferricyanide and hydrogen peroxide were examined at the CNT-modified electrodes. Electrodes modified with SWCNTs usually had better electron-transfer properties than MWCNT-modified electrodes. Glucose biosensors were also prepared with electropolymerized polyphenylenediamine films, CNT materials, and glucose oxidase. Amperometric behavior in glucose determination was examined. SWCNT-modified glucose biosensors usually had a wider dynamic range (from 0.1 to 5.5 mmol L−1) and greater sensitivity in glucose determination. The detection limit was estimated to be 0.05 mmol L−1.
Keywords: Carbon nanotubes; Tube internal diameter; Electron-transfer properties; Polyphenylenediamine; Glucose biosensors
Spectroscopic study of the interaction of actinomycin D with oligonucleotides carrying the central base sequences -XGCY- and -XGGCCY- using multivariate methods by M. Vives; R. Tauler; R. Eritja; R. Gargallo (pp. 311-320).
The interactions of actinomycin D (ACTD) with the oligonucleotides 5′-CAAAGCTTTG-3′, 5′-CATGGCCATG-3′ and 5′-TATGGCCATA-3′ were investigated by means of acid–base titrations and mole-ratio and melting experiments monitored by molecular absorption and circular dichroism (CD) spectroscopies. For each experiment, CD and molecular absorption spectra were recorded at each point in the experiment, and later analyzed via appropriate multivariate data analysis methods. The study of the interactions between these oligonucleotides and ACTD at 25 °C showed the formation of an interaction complex with a stoichiometry of 1:1 (ACTD:duplex) and values for the log(formation constant) of 5.1 ± 0.3, 6.4 ± 0.2, and 5.6 ± 0.2, respectively. An additional interaction complex at higher temperatures was also detected, which might be related to the single-stranded forms of the oligonucleotides.
Keywords: Actinomycin D; Multivariate analysis; Oligonucleotides; Spectroscopy; Multivariate curve resolution
Electron-impact and glow-discharge ionization LC–MS analysis of green tea tincture by Jacob L. Venzie; Joaudimir Castro; M. V. Balarama Krishna; Dwella M. Nelson; R. Kenneth Marcus (pp. 321-333).
A liquid chromatography–particle-beam mass spectrometer (LC–PB/MS) with interchangeable electron-impact (EI) and glow-discharge (GD) ion sources was evaluated for future application in analysis of botanical extracts. In this work a green tea tincture was characterized for a series of catechin components (catechin, epicatechin, epigallocatechin, and epigallocatechin gallate (EGCG)) and caffeine. Special emphasis was given to EGCG and caffeine, because they are important in determining the possible health effects of the green tea. The effects of instrument operating conditions were evaluated for the EI and GD ionization sources to determine their effect on analyte intensities and fragmentation patterns. These studies furnished information about the effects of these conditions in determining possible ionization pathways in the two ion sources. The mass spectra of these compounds obtained with the GD ion source are EI-like in appearance, with clearly identified molecular ions and fragmentation patterns that are easily rationalized. The absolute limits of detection for EGCG and caffeine were, respectively, 11 ng and 0.77 ng for the EI source and 3.2 ng and 0.61 ng for the GD source. The PB/EIMS and PB/GDMS combinations can be operated in a flow-injection mode, wherein the analyte is injected directly into the mobile phase, or coupled to high-performance liquid chromatography (HPLC), enabling LC–MS analysis of complex mixtures. A reversed-phase chromatographic separation of the green tea tincture was performed on a commercial C18 column using a gradient of water (containing 0.1% TFA) and ACN. Quantification of EGCG and caffeine was performed by the standard addition method. The amounts of EGCG and caffeine in the tested green tea tincture were each ∼14 mg mL−1.
Keywords: Dietary supplements; Catechins; Particle beam; Electron impact; Glow discharge; Mass spectrometry
Microanalyzer for biomonitoring lead (Pb) in blood and urine by Wassana Yantasee; Charles Timchalk; Yuehe Lin (pp. 335-341).
Biomonitoring of lead (Pb) in blood and urine enables quantitative evaluation of human occupational and environmental exposures to Pb. State-of-the-art ICP–MS instruments can only analyze metals in laboratories, resulting in lengthy turnaround times, and they are expensive. In response to the growing need for a metal analyzer capable of on-site, real-time monitoring of trace toxic metals in individuals, we developed a portable microanalyzer based on flow-injection/stripping voltammetry (ASV), and validated the system using rat blood and urine spiked with known amounts of Pb. Fouling of electrodes by proteins often prevents the effective use of electrochemical sensors in biological matrices. Minimization of such fouling was accomplished with suitable sample pretreatment and by establishing turbulent flow of blood and urine containing Pb onto the electrode inside the microanalyzer, which resulted in no apparent electrode fouling even when the samples contained 50% urine or 10% blood by volume. No matrix effect was observed for the voltammetric Pb signals, even when the samples contained 10% blood or 10% urine. The microanalyzer offered linear concentration ranges relevant to Pb exposure levels in humans (0–20 ppb in 10% blood samples, 0–50 ppb in 50% urine samples). The device showed excellent sensitivity and reproducibility; Pb detection limits were 0.44 ppb and 0.46 ppb, and % R.S.D. was 4.9 and 2.4 in 50% urine and 10% blood samples, respectively. It gave similar Pb concentrations in blood and urine to those measured by ICP–MS. It offered high throughput (3 min per sample) and economical use of samples (60 μL per measurement) as well as low reagent consumption (1 μg of Hg per measurement), thus minimizing environmental concerns associated with mercury use. Since it is miniaturized, the microanalyzer is portable and field-deployable. Thus, it shows much promise as the next-generation analyzer for the biomonitoring of toxic metals.
Keywords: Biomonitoring; Pb; Flow injection; Stripping voltammetry; Urine; Blood
Determination of 90Sr in contaminated environmental samples by tuneable bandpass dynamic reaction cell ICP–MS by V. F. Taylor; R. D. Evans; R. J. Cornett (pp. 343-350).
A rapid method for the extraction and determination of 90Sr in natural water, plant and sediment samples was developed using extraction chromatography and dynamic reaction cell ICP–MS, with O2 as a reaction gas. While isobaric interference from the stable isotope 90Zr was efficiently removed by this method, interferences produced from in-cell reactions with Fe+ and Ni+ required suppression by tuneable bandpass, and in sediments, additional chromatographic separation. Method detection limits were 0.1 pg g−1 (0.5 Bq g−1), 0.04 pg g−1(0.2 Bq g−1), and 3 pg L−1 (5 Bq L−1) for sediments, plant and water samples, respectively, and 90Sr concentrations determined by ICP–MS were in good agreement with activities determined by Cerenkov counting and with certified reference values. While mass spectrometric determination does not rival detection limits achievable by radiometric counting, radiometric determination of 90Sr, a pure beta-emitter, is hindered by long analysis times (several weeks); the comparatively fast analysis achieved via ICP–MS enables same-day preparation and analysis of samples, making this an important technique for the environmental monitoring of areas contaminated by radioactivity.
Keywords: Strontium 90; Fission products; Reaction cell inductively coupled plasma mass spectrometry; Tuneable bandpass; Gas phase reaction; Rapid assessment protocol
Application of an in-line imprinted polymer column in a potentiometric flow-injection chemical sensor to the determination of the carbamate pesticide carbaryl in complex biological matrices by Jamil Hantash; Alan Bartlett; Philip Oldfield; Georges Dénès; Roger O’Rielly; Francois David (pp. 351-357).
A flow-injection biosensor-like system based on a nonenzymatic approach has been developed to determine the carbamate pesticide carbaryl in complex biological samples without lengthy and expensive extraction steps. Molecularly imprinted polymeric beads were used to immobilize carbaryl from biological samples. pH variation permitted the elution of carbaryl from the binding cavity to the flow cell. A pH electrode was used to detect changes in the charge of carbaryl in the sample solution resulting from the protonation and deprotonation of the molecule over different pH ranges. At pH 2.0, the secondary amine group is protonated, giving a (+1) charge to the carbaryl molecule. At pH 8.0, the ionized carbaryl loses a proton to become neutral, changing the local pH of the flow cell. The pH change at the flow cell generated by the deprotonation of carbaryl ion in alkaline medium was used to determine the carbaryl concentration. Parameters influencing the performance of the system were optimized for use in the detection procedure. The validated biosensor-like system had a carbaryl detection limit of 10.0 μg/mL and a response that was linear (r 2 > 0.98) over the concentration range of 10.0–00 μg/mL.
Keywords: Pesticides; Potentiometric; Flow injection; Biosensor-like; Molecularly imprinted polymer; Suspension polymerization; Detection
Analysis of trace levels of pesticides in rainwater by SPME and GC-tandem mass spectrometry after derivatisation with PFFBr by Anne Scheyer; Olivier Briand; Stéphane Morville; Philippe Mirabel; Maurice Millet (pp. 359-368).
Solid-phase microextraction (SPME) was used for the analysis of some pesticides (bromoxynil, chlorotoluron, diuron, isoproturon, 2,4-MCPA, MCPP and 2,4-D) in rainwater after derivatisation with PFBBr and gas chromatography-ion trap mass spectrometry. The derivatisation procedure was optimized by testing different methods: direct derivatisation in the aqueous phase followed by SPME extraction, on-fibre derivatisation and derivatisation in the injector. The best result was obtained by headspace coating the PDMS/DVB fibre with PFBBr for 10 min followed by direct SPME extraction for 60 min at 68 °C (pH 2 and 75% NaCl). Good detection limits were obtained for all the compounds: these ranged between 10 and 1,000 ng L−1 with a relatively high uncertainty due to the combination of derivatisation and SPME extraction steps. The optimized procedure was applied to the analysis of pesticides in rainwater and results obtained shows that this method is a fast and simple technique to assess the spatial and temporal variations of concentrations of pesticides in rainwater.
Keywords: SPME; PFBBr derivatisation; GC-MSMS; Rainwater