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Analytical and Bioanalytical Chemistry (v.395, #3)


Microdischarges for analytical applications by Joachim Franzke (pp. 547-548).
is Director of the Department of Miniaturization at ISAS. His research interests are related to the development of methods and miniaturized instrumentation for discharge-based analytical spectrometry.

Microdischarges for analytical applications by Joachim Franzke (pp. 547-548).
is Director of the Department of Miniaturization at ISAS. His research interests are related to the development of methods and miniaturized instrumentation for discharge-based analytical spectrometry.

The micro-discharge family (dark, corona, and glow-discharge) for analytical applications realized by dielectric barriers by J. Franzke (pp. 549-557).
The similarity principles of electric plasmas, and the current-voltage characteristics of the most prominent kinds of discharges used for analytical applications, are discussed. Most of the discharges can be miniaturized, and some of the analytical applications of different discharges can be realized by use of dielectric barriers for analytical applications, for example element spectrometry, as an ionization source for ion-mobility spectrometry or organic mass spectrometry, and as an electrospray ionization source.

Keywords: Dielectric barriers; Discharges; Electric plasmas


The micro-discharge family (dark, corona, and glow-discharge) for analytical applications realized by dielectric barriers by J. Franzke (pp. 549-557).
The similarity principles of electric plasmas, and the current-voltage characteristics of the most prominent kinds of discharges used for analytical applications, are discussed. Most of the discharges can be miniaturized, and some of the analytical applications of different discharges can be realized by use of dielectric barriers for analytical applications, for example element spectrometry, as an ionization source for ion-mobility spectrometry or organic mass spectrometry, and as an electrospray ionization source.

Keywords: Dielectric barriers; Discharges; Electric plasmas


Exploring microdischarges for portable sensing applications by Y. B. Gianchandani; S. A. Wright; C. K. Eun; C. G. Wilson; B. Mitra (pp. 559-575).
This paper describes the use of microdischarges as transducing elements in sensors and detectors. Chemical and physical sensing of gases, chemical sensing of liquids, and radiation detection are described. These applications are explored from the perspective of their use in portable microsystems, with emphasis on compactness, power consumption, the ability to operate at or near atmospheric pressure (to reduce pumping challenges), and the ability to operate in an air ambient (to reduce the need for reservoirs of carrier gases). Manufacturing methods and performance results are described for selected examples. Figure Side-view photograph of an ultraviolet light source that uses microdischarges

Keywords: Plasma; Discharge; Chemical; Radiation; Pressure


Exploring microdischarges for portable sensing applications by Y. B. Gianchandani; S. A. Wright; C. K. Eun; C. G. Wilson; B. Mitra (pp. 559-575).
This paper describes the use of microdischarges as transducing elements in sensors and detectors. Chemical and physical sensing of gases, chemical sensing of liquids, and radiation detection are described. These applications are explored from the perspective of their use in portable microsystems, with emphasis on compactness, power consumption, the ability to operate at or near atmospheric pressure (to reduce pumping challenges), and the ability to operate in an air ambient (to reduce the need for reservoirs of carrier gases). Manufacturing methods and performance results are described for selected examples. Figure Side-view photograph of an ultraviolet light source that uses microdischarges

Keywords: Plasma; Discharge; Chemical; Radiation; Pressure


Helium–hydrogen microplasma device (MPD) on postage-stamp-size plastic–quartz chips by Scott Weagant; Vassili Karanassios (pp. 577-589).
A new design of a miniaturized, atmospheric-pressure, low-power (e.g., battery-operated), self-igniting, planar-geometry microplasma device (MPD) for use with liquid microsamples is described. The inexpensive MPD was a hybrid, three-substrate quartz–plastic–plastic structure and it was formed on chips with area the size of a small postage stamp. The substrates were chosen for rapid prototyping and for speedy device-geometry testing and evaluation. The ~700-µm (diameter) and 7-mm (long) He–H2 (3% H2) microplasma was formed by applying high-voltage ac between two needle electrodes. Operating conditions were found to be critical in sustaining stable microplasma on plastic substrates. Spectral interference from the electrode materials was not observed. A small-size, electrothermal vaporization system was used for introduction of microliter volumes of liquids into the MPD. The microplasma was operated from an inexpensive power supply. And, operation from a 14.4-V battery has been demonstrated. Microplasma background emission in the spectral range between 200 and 850 nm obtained using a portable, fiber-optic spectrometer is reported. Analyte emission from microliter volumes of dilute single-element standard solutions of Cd, Cu, K, Li, Mg, Mn, Na, Pb, and Zn is documented. Element-dependent precision was between 10–25% (the average was 15%) and detection limits ranged between 1.5 and 350 ng. The system was used for the determination of Na in diluted bottled-water samples.

Keywords: Microplasma (MPD); Liquid microsamples; Optical emission spectrometry; Plasma instrumentation; Electrothermal vaporization (ETV)


Helium–hydrogen microplasma device (MPD) on postage-stamp-size plastic–quartz chips by Scott Weagant; Vassili Karanassios (pp. 577-589).
A new design of a miniaturized, atmospheric-pressure, low-power (e.g., battery-operated), self-igniting, planar-geometry microplasma device (MPD) for use with liquid microsamples is described. The inexpensive MPD was a hybrid, three-substrate quartz–plastic–plastic structure and it was formed on chips with area the size of a small postage stamp. The substrates were chosen for rapid prototyping and for speedy device-geometry testing and evaluation. The ~700-µm (diameter) and 7-mm (long) He–H2 (3% H2) microplasma was formed by applying high-voltage ac between two needle electrodes. Operating conditions were found to be critical in sustaining stable microplasma on plastic substrates. Spectral interference from the electrode materials was not observed. A small-size, electrothermal vaporization system was used for introduction of microliter volumes of liquids into the MPD. The microplasma was operated from an inexpensive power supply. And, operation from a 14.4-V battery has been demonstrated. Microplasma background emission in the spectral range between 200 and 850 nm obtained using a portable, fiber-optic spectrometer is reported. Analyte emission from microliter volumes of dilute single-element standard solutions of Cd, Cu, K, Li, Mg, Mn, Na, Pb, and Zn is documented. Element-dependent precision was between 10–25% (the average was 15%) and detection limits ranged between 1.5 and 350 ng. The system was used for the determination of Na in diluted bottled-water samples.

Keywords: Microplasma (MPD); Liquid microsamples; Optical emission spectrometry; Plasma instrumentation; Electrothermal vaporization (ETV)


Rapid screening of active ingredients in drugs by mass spectrometry with low-temperature plasma probe by Yueying Liu; Ziqing Lin; Sichun Zhang; Chengdui Yang; Xinrong Zhang (pp. 591-599).
A high-throughput method for rapid screening of active ingredients in drugs has been developed with mass spectrometry coupled to a low-temperature plasma (LTP) probe ion source. Without sample preparation or pretreatment, the active ingredients of 11 types of commercial pharmaceuticals, including hormones, antipyretic analgesics, cardiovascular, digestant, neuro-psychotherapeutic, diuretic, antithyroid, sulfa anti-inflammatory, antiparastic, sedative-hypnotics, and antibacterial, were directly desorbed/ionized and detected by a linear ion trap mass spectrometry (MS). The structures of these ingredients were elucidated by tandem MS. The analysis of 18 methyltestosterone tablets could be accomplished within 1.9 min, which allows fast detection with a speed of approximate 600 samples within 1 h. This work demonstrated that LTP probe ion source combined with MS is a high-throughput method for screening of pharmaceuticals and potentially applied to on-line quality control in pharmaceutical industry. Figure Schematic diagram of LTP probe for ambient ionization MS

Keywords: Rapid screening; LTP probe; Drugs; Mass spectrometry; Ambient ionization


Rapid screening of active ingredients in drugs by mass spectrometry with low-temperature plasma probe by Yueying Liu; Ziqing Lin; Sichun Zhang; Chengdui Yang; Xinrong Zhang (pp. 591-599).
A high-throughput method for rapid screening of active ingredients in drugs has been developed with mass spectrometry coupled to a low-temperature plasma (LTP) probe ion source. Without sample preparation or pretreatment, the active ingredients of 11 types of commercial pharmaceuticals, including hormones, antipyretic analgesics, cardiovascular, digestant, neuro-psychotherapeutic, diuretic, antithyroid, sulfa anti-inflammatory, antiparastic, sedative-hypnotics, and antibacterial, were directly desorbed/ionized and detected by a linear ion trap mass spectrometry (MS). The structures of these ingredients were elucidated by tandem MS. The analysis of 18 methyltestosterone tablets could be accomplished within 1.9 min, which allows fast detection with a speed of approximate 600 samples within 1 h. This work demonstrated that LTP probe ion source combined with MS is a high-throughput method for screening of pharmaceuticals and potentially applied to on-line quality control in pharmaceutical industry. Figure Schematic diagram of LTP probe for ambient ionization MS

Keywords: Rapid screening; LTP probe; Drugs; Mass spectrometry; Ambient ionization


Plasma stencilling methods for cell patterning by Jean-Philippe Frimat; Heike Menne; Antje Michels; Silke Kittel; Raffael Kettler; Sabine Borgmann; Joachim Franzke; Jonathan West (pp. 601-609).
In this paper we describe plasma stencilling techniques for patterning 10 mammalian cell lines on hydrophobic and cell repellent poly(dimethylsiloxane) (PDMS), methylated glass and bacterial grade polystyrene surfaces. An air plasma produced with a Tesla generator operating at atmospheric pressure was used with microengineered stencils for patterned surface oxidation, selectively transforming the surface to a hydrophilic state to enable cell adhesion and growth. Plasma stencilling obviates the need for directly patterning cell adhesion molecules. Instead, during cell culture, adhesion proteins from the media assemble in a bioactive form on the hydrophilic regions. Critically, the removal of protein patterning prior to cell culture provides the option to also use PDMS–PDMS plasma bonding to incorporate cell patterns within microfluidic systems. Linear patterns were generated using PDMS microchannel stencils, and polyimide stencils with through holes were used for the production of cellular arrays. For the production of smaller cellular arrays, a novel microcapillary-based dielectric barrier discharge system was developed. A numerical method to characterise the cell patterns is also introduced and was used to demonstrate that plasma stencilling is highly effective, with complete patterns confined during long term cell culture (>10 days). In summary, plasma stencilling is simple, rapid, inexpensive, reproducible and a potentially universal cell line patterning capability. Figure Microfluidic plasma stencilling for generating cell lines.

Keywords: Cell patterning; Plasmas; Stencil; Poly(dimethylsiloxane); Dielectric barrier discharge; Microfluidics


Plasma stencilling methods for cell patterning by Jean-Philippe Frimat; Heike Menne; Antje Michels; Silke Kittel; Raffael Kettler; Sabine Borgmann; Joachim Franzke; Jonathan West (pp. 601-609).
In this paper we describe plasma stencilling techniques for patterning 10 mammalian cell lines on hydrophobic and cell repellent poly(dimethylsiloxane) (PDMS), methylated glass and bacterial grade polystyrene surfaces. An air plasma produced with a Tesla generator operating at atmospheric pressure was used with microengineered stencils for patterned surface oxidation, selectively transforming the surface to a hydrophilic state to enable cell adhesion and growth. Plasma stencilling obviates the need for directly patterning cell adhesion molecules. Instead, during cell culture, adhesion proteins from the media assemble in a bioactive form on the hydrophilic regions. Critically, the removal of protein patterning prior to cell culture provides the option to also use PDMS–PDMS plasma bonding to incorporate cell patterns within microfluidic systems. Linear patterns were generated using PDMS microchannel stencils, and polyimide stencils with through holes were used for the production of cellular arrays. For the production of smaller cellular arrays, a novel microcapillary-based dielectric barrier discharge system was developed. A numerical method to characterise the cell patterns is also introduced and was used to demonstrate that plasma stencilling is highly effective, with complete patterns confined during long term cell culture (>10 days). In summary, plasma stencilling is simple, rapid, inexpensive, reproducible and a potentially universal cell line patterning capability. Figure Microfluidic plasma stencilling for generating cell lines.

Keywords: Cell patterning; Plasmas; Stencil; Poly(dimethylsiloxane); Dielectric barrier discharge; Microfluidics


Electrical generators driving microhollow and dielectric barrier discharges applied for analytical chemistry by R. Heming; A. Michels; S. B. Olenici; S. Tombrink; J. Franzke (pp. 611-618).
Scaling down the size of plasma discharges would reduce the amount of gases, liquids, and consumables required, which in turn would decrease the operating costs. Nevertheless, the application of a specialized plasma generator for microhollow cathode discharges (MHCD) and dielectric barrier discharges are driven with commercially available power sources. Those generators are bulky and expensive and their overall efficiency is poor. This work develops and explains several circuit topologies and design hints to excite MHCD and dielectric barrier discharge (DBD) plasmas with respect to its system with as low as possible input power in a very efficient way. Benefits in sensitivity and life expectancy are shown. The generator for the MHCD needs voltages up to 7 V and consumes up to 5 W. The DBD generator has an input power of 3 W and produces a fast rising output pulse up to 9 kV, which has a time duration of 2 µs. These low-power circuits offer the operation with batteries.

Keywords: Plasma generators; Microhollow cathode discharge (MHCD); Dielectric barrier discharge (DBD); Atmospheric pressure glow discharge (APGD); Radio-frequency capacitive coupled plasma (rf-CCP)


Electrical generators driving microhollow and dielectric barrier discharges applied for analytical chemistry by R. Heming; A. Michels; S. B. Olenici; S. Tombrink; J. Franzke (pp. 611-618).
Scaling down the size of plasma discharges would reduce the amount of gases, liquids, and consumables required, which in turn would decrease the operating costs. Nevertheless, the application of a specialized plasma generator for microhollow cathode discharges (MHCD) and dielectric barrier discharges are driven with commercially available power sources. Those generators are bulky and expensive and their overall efficiency is poor. This work develops and explains several circuit topologies and design hints to excite MHCD and dielectric barrier discharge (DBD) plasmas with respect to its system with as low as possible input power in a very efficient way. Benefits in sensitivity and life expectancy are shown. The generator for the MHCD needs voltages up to 7 V and consumes up to 5 W. The DBD generator has an input power of 3 W and produces a fast rising output pulse up to 9 kV, which has a time duration of 2 µs. These low-power circuits offer the operation with batteries.

Keywords: Plasma generators; Microhollow cathode discharge (MHCD); Dielectric barrier discharge (DBD); Atmospheric pressure glow discharge (APGD); Radio-frequency capacitive coupled plasma (rf-CCP)


Microfluidics for bioapplications by Oliver Geschke (pp. 619-619).
is Associate Professor at the Department of Micro- and Nanotechnology (DTU Nanotech) at the Technical University of Denmark. He was educated as an analytical chemist and his research interest is now microfabrication techniques for inexpensive polymer microsystems, mainly for biological and biotechnological applications. Direct prototyping such as laser ablation and micromilling and reproduction methods such as hot/soft embossing and molding in a variety of polymer materials are the techniques commonly applied in his laboratory. Oliver has been the chief editor of the textbook Microsystems engineering of lab-on-a-chip devices and provides courses on this subject at various international conferences. His research group currently consists of one postdoctoral fellow and four PhD students.

Microfluidics for bioapplications by Oliver Geschke (pp. 619-619).
is Associate Professor at the Department of Micro- and Nanotechnology (DTU Nanotech) at the Technical University of Denmark. He was educated as an analytical chemist and his research interest is now microfabrication techniques for inexpensive polymer microsystems, mainly for biological and biotechnological applications. Direct prototyping such as laser ablation and micromilling and reproduction methods such as hot/soft embossing and molding in a variety of polymer materials are the techniques commonly applied in his laboratory. Oliver has been the chief editor of the textbook Microsystems engineering of lab-on-a-chip devices and provides courses on this subject at various international conferences. His research group currently consists of one postdoctoral fellow and four PhD students.

Emerging optofluidic technologies for point-of-care genetic analysis systems: a review by Desmond Brennan; John Justice; Brian Corbett; Tommie McCarthy; Paul Galvin (pp. 621-636).
This review describes recently emerging optical and microfluidic technologies suitable for point-of-care genetic analysis systems. Such systems must rapidly detect hundreds of mutations from biological samples with low DNA concentration. We review optical technologies delivering multiplex sensitivity and compatible with lab-on-chip integration for both tagged and non-tagged optical detection, identifying significant source and detector technology emerging from telecommunications technology. We highlight the potential for improved hybridization efficiency through careful microfluidic design and outline some novel enhancement approaches using target molecule confinement. Optimization of fluidic parameters such as flow rate, channel height and time facilitates enhanced hybridization efficiency and consequently detection performance as compared with conventional assay formats (e.g. microwell plates). We highlight lab-on-chip implementations with integrated microfluidic control for “sample-to-answer” systems where molecular biology protocols to realize detection of target DNA sequences from whole blood are required. We also review relevant technology approaches to optofluidic integration, and highlight the issue of biomolecule compatibility. Key areas in the development of an integrated optofluidic system for DNA hybridization are optical/fluidic integration and the impact on biomolecules immobilized within the system. A wide range of technology platforms have been advanced for detection, quantification and other forms of characterization of a range of biomolecules (e.g. RNA, DNA, protein and whole cell). Owing to the very different requirements for sample preparation, manipulation and detection of the different types of biomolecules, this review is focused primarily on DNA–DNA interactions in the context of point-of-care analysis systems.

Keywords: Microfluidics; Optical detection; DNA; Surface chemistry


Emerging optofluidic technologies for point-of-care genetic analysis systems: a review by Desmond Brennan; John Justice; Brian Corbett; Tommie McCarthy; Paul Galvin (pp. 621-636).
This review describes recently emerging optical and microfluidic technologies suitable for point-of-care genetic analysis systems. Such systems must rapidly detect hundreds of mutations from biological samples with low DNA concentration. We review optical technologies delivering multiplex sensitivity and compatible with lab-on-chip integration for both tagged and non-tagged optical detection, identifying significant source and detector technology emerging from telecommunications technology. We highlight the potential for improved hybridization efficiency through careful microfluidic design and outline some novel enhancement approaches using target molecule confinement. Optimization of fluidic parameters such as flow rate, channel height and time facilitates enhanced hybridization efficiency and consequently detection performance as compared with conventional assay formats (e.g. microwell plates). We highlight lab-on-chip implementations with integrated microfluidic control for “sample-to-answer” systems where molecular biology protocols to realize detection of target DNA sequences from whole blood are required. We also review relevant technology approaches to optofluidic integration, and highlight the issue of biomolecule compatibility. Key areas in the development of an integrated optofluidic system for DNA hybridization are optical/fluidic integration and the impact on biomolecules immobilized within the system. A wide range of technology platforms have been advanced for detection, quantification and other forms of characterization of a range of biomolecules (e.g. RNA, DNA, protein and whole cell). Owing to the very different requirements for sample preparation, manipulation and detection of the different types of biomolecules, this review is focused primarily on DNA–DNA interactions in the context of point-of-care analysis systems.

Keywords: Microfluidics; Optical detection; DNA; Surface chemistry


New directions in medical biosensors employing poly(3,4-ethylenedioxy thiophene) derivative-based electrodes by Noemi Rozlosnik (pp. 637-645).
Demand is growing in the field of medical diagnostics for simple, disposable devices that also demonstrate fast response times, are easy to handle, are cost-efficient, and are suitable for mass production. Polymer-based microfluidic devices meet the requirements of cost efficiency and mass production and they are suitable for biosensor applications. Conducting polymer-based electrochemical sensors have shown numerous advantages in a number of areas related to human health, such as the diagnosis of infectious diseases, genetic mutations, drug discovery, forensics and food technology, due to their simplicity and high sensitivity. One of the most promising group of conductive polymers is poly(3,4-ethylenedioxythiophene) (PEDOT) and its derivatives due to their attractive properties: high stability, high conductivity (up to 400–600 S/cm) and high transparency. This review paper summarizes newly developed methods associated with the application of PEDOT to diagnostic sensing.

Keywords: Biosensor; Conductive polymer; PEDOT; Electrochemistry; Microfabrication


New directions in medical biosensors employing poly(3,4-ethylenedioxy thiophene) derivative-based electrodes by Noemi Rozlosnik (pp. 637-645).
Demand is growing in the field of medical diagnostics for simple, disposable devices that also demonstrate fast response times, are easy to handle, are cost-efficient, and are suitable for mass production. Polymer-based microfluidic devices meet the requirements of cost efficiency and mass production and they are suitable for biosensor applications. Conducting polymer-based electrochemical sensors have shown numerous advantages in a number of areas related to human health, such as the diagnosis of infectious diseases, genetic mutations, drug discovery, forensics and food technology, due to their simplicity and high sensitivity. One of the most promising group of conductive polymers is poly(3,4-ethylenedioxythiophene) (PEDOT) and its derivatives due to their attractive properties: high stability, high conductivity (up to 400–600 S/cm) and high transparency. This review paper summarizes newly developed methods associated with the application of PEDOT to diagnostic sensing.

Keywords: Biosensor; Conductive polymer; PEDOT; Electrochemistry; Microfabrication


Miniaturized tools and devices for bioanalytical applications: an overview by Michal Chudy; Ilona Grabowska; Patrycja Ciosek; Alicja Filipowicz-Szymanska; Dorota Stadnik; Iwona Wyzkiewicz; Elzbieta Jedrych; Marcin Juchniewicz; Maciej Skolimowski; Karina Ziolkowska; Radoslaw Kwapiszewski (pp. 647-668).
This article presents an overview of various miniaturized devices and technologies developed by our group. Innovative, fast and cheap procedures for the fabrication of laboratory microsystems based on commercially available materials are reported and compared with well-established microfabrication techniques. The modules fabricated and tested in our laboratory can be used independently or they can be set up in different configurations to form functional measurement systems. We also report further applications of the presented modules e.g. disposable poly(dimethylsiloxane) (PDMS) microcuvettes, fibre optic detectors, potentiometric sensors platforms, microreactors and capillary electrophoresis (CE) microchips as well as integrated microsystems e.g. double detection microanalytical systems, devices for studying enzymatic reactions and a microsystem for cell culture and lysis.

Keywords: Capillary film; Bonding-less (b-less) and pull-out techniques; Enzymatic microreactors; Microchips for capillary electrophoresis (MCE); Dual detection; Cell culture passage and lysis


Miniaturized tools and devices for bioanalytical applications: an overview by Michal Chudy; Ilona Grabowska; Patrycja Ciosek; Alicja Filipowicz-Szymanska; Dorota Stadnik; Iwona Wyzkiewicz; Elzbieta Jedrych; Marcin Juchniewicz; Maciej Skolimowski; Karina Ziolkowska; Radoslaw Kwapiszewski (pp. 647-668).
This article presents an overview of various miniaturized devices and technologies developed by our group. Innovative, fast and cheap procedures for the fabrication of laboratory microsystems based on commercially available materials are reported and compared with well-established microfabrication techniques. The modules fabricated and tested in our laboratory can be used independently or they can be set up in different configurations to form functional measurement systems. We also report further applications of the presented modules e.g. disposable poly(dimethylsiloxane) (PDMS) microcuvettes, fibre optic detectors, potentiometric sensors platforms, microreactors and capillary electrophoresis (CE) microchips as well as integrated microsystems e.g. double detection microanalytical systems, devices for studying enzymatic reactions and a microsystem for cell culture and lysis.

Keywords: Capillary film; Bonding-less (b-less) and pull-out techniques; Enzymatic microreactors; Microchips for capillary electrophoresis (MCE); Dual detection; Cell culture passage and lysis


Increasing the specificity and function of DNA microarrays by processing arrays at different stringencies by Martin Dufva; Jesper Petersen; Lena Poulsen (pp. 669-677).
DNA microarrays have for a decade been the only platform for genome-wide analysis and have provided a wealth of information about living organisms. DNA microarrays are processed today under one condition only, which puts large demands on assay development because all probes on the array need to function optimally under one condition only. Microarrays are often burdened with a significant degree of cross-hybridization, because of a poor combination of assay conditions and probe choice. As reviewed here, a number of promising microfluidics-based technologies can provide automatic processing of arrays under different assay conditions. These new array processors provide researchers and assay developers with novel possibilities to construct highly specific DNA arrays even towards regions of DNA greatly varying in G + C content. These array processors are also a powerful development tool for building arrays, because they combine high sample throughput with investigation of optimal assay conditions. The array processors can increase specificity in all DNA microarray assays, e.g. for gene expression, and microRNA and mutation analysis. Increased specificity of the array will also benefit microarray-based loci selection prior to high-throughput sequencing.

Keywords: Microarray; DNA sequencing; Nearest neighbor method; Genotyping; Diagnostics; Gradient; Fluidics


Increasing the specificity and function of DNA microarrays by processing arrays at different stringencies by Martin Dufva; Jesper Petersen; Lena Poulsen (pp. 669-677).
DNA microarrays have for a decade been the only platform for genome-wide analysis and have provided a wealth of information about living organisms. DNA microarrays are processed today under one condition only, which puts large demands on assay development because all probes on the array need to function optimally under one condition only. Microarrays are often burdened with a significant degree of cross-hybridization, because of a poor combination of assay conditions and probe choice. As reviewed here, a number of promising microfluidics-based technologies can provide automatic processing of arrays under different assay conditions. These new array processors provide researchers and assay developers with novel possibilities to construct highly specific DNA arrays even towards regions of DNA greatly varying in G + C content. These array processors are also a powerful development tool for building arrays, because they combine high sample throughput with investigation of optimal assay conditions. The array processors can increase specificity in all DNA microarray assays, e.g. for gene expression, and microRNA and mutation analysis. Increased specificity of the array will also benefit microarray-based loci selection prior to high-throughput sequencing.

Keywords: Microarray; DNA sequencing; Nearest neighbor method; Genotyping; Diagnostics; Gradient; Fluidics


Application of microbioreactors in fermentation process development: a review by Daniel Schäpper; Muhd Nazrul Hisham Zainal Alam; Nicolas Szita; Anna Eliasson Lantz; Krist V. Gernaey (pp. 679-695).
Biotechnology process development involves strain testing and improvement steps aimed at increasing yields and productivity. This necessitates the high-throughput screening of many potential strain candidates, a task currently mainly performed in shake flasks or microtiter plates. However, these methods have some drawbacks, such as the low data density (usually only end-point measurements) and the lack of control over cultivation conditions in standard shake flasks. Microbioreactors can offer the flexibility and controllability of bench-scale reactors and thus deliver results that are more comparable to large-scale fermentations, but with the additional advantages of small size, availability of online cultivation data and the potential for automation. Current microbioreactor technology is analyzed in this review paper, focusing on its industrial applicability, and directions for future research are presented.

Keywords: Microbioreactor; Fermentation; Bioprocess monitoring; Microfluidic suspension culture


Application of microbioreactors in fermentation process development: a review by Daniel Schäpper; Muhd Nazrul Hisham Zainal Alam; Nicolas Szita; Anna Eliasson Lantz; Krist V. Gernaey (pp. 679-695).
Biotechnology process development involves strain testing and improvement steps aimed at increasing yields and productivity. This necessitates the high-throughput screening of many potential strain candidates, a task currently mainly performed in shake flasks or microtiter plates. However, these methods have some drawbacks, such as the low data density (usually only end-point measurements) and the lack of control over cultivation conditions in standard shake flasks. Microbioreactors can offer the flexibility and controllability of bench-scale reactors and thus deliver results that are more comparable to large-scale fermentations, but with the additional advantages of small size, availability of online cultivation data and the potential for automation. Current microbioreactor technology is analyzed in this review paper, focusing on its industrial applicability, and directions for future research are presented.

Keywords: Microbioreactor; Fermentation; Bioprocess monitoring; Microfluidic suspension culture


Biomimetic membranes for sensor and separation applications by Claus Hélix Nielsen (pp. 697-718).
Biological membranes constitute the set of membranes defining boundaries and organelles in living cells—the structural and functional building blocks of all known living organisms. The integrity of the cell depends on its ability to separate inside from outside and yet at the same time allow massive transport of matter in and out the cell. Nature has elegantly met this challenge by developing membranes in the form of lipid bilayers in which specialized and highly efficient transport proteins are incorporated. This raises the question: is it possible to mimic biological membranes and create membrane-based sensor and/or separation devices? In the development of biomimetic sensor/separation technology, both channels (ion and water channels) and carriers (transporters) are important. Generally, each class of transport proteins conducts specific molecular species in and out of the cell while preventing the passage of others, a property critical for the overall conservation of the cells internal pH and salt concentration. Both ion and water channels are highly efficient membrane pore proteins capable of transporting solutes at very high rates, up to 109 molecules per second. Carrier proteins generally have a lower turnover but are capable of transport against gradients. For both classes of proteins, their unique flux-properties make them interesting as candidates in biomimetic sensor/separation devices. An ideal sensor/separation device requires the supporting biomimetic matrix to be virtually impermeable to anything but the solute in question. In practice, however, a biomimetic support matrix will generally have finite permeabilities to water, electrolytes, and non-electrolytes. The feasibility of a biomimetic device thus depends on the relative transport contribution from both protein and biomimetic support matrix. Also the stability of the incorporated protein must be addressed and the protein-biomimetic matrix must be encapsulated in order to protect it and make it sufficiently stable in a final application. Here I will review and discuss these challenges and how they are met in some current developments of biomimetic sensor/separation devices.

Keywords: Biomaterials; Biopolymers/lipids; Biosensors; Microfluidics/microfabrication; Separations/instrumentation; Water


Biomimetic membranes for sensor and separation applications by Claus Hélix Nielsen (pp. 697-718).
Biological membranes constitute the set of membranes defining boundaries and organelles in living cells—the structural and functional building blocks of all known living organisms. The integrity of the cell depends on its ability to separate inside from outside and yet at the same time allow massive transport of matter in and out the cell. Nature has elegantly met this challenge by developing membranes in the form of lipid bilayers in which specialized and highly efficient transport proteins are incorporated. This raises the question: is it possible to mimic biological membranes and create membrane-based sensor and/or separation devices? In the development of biomimetic sensor/separation technology, both channels (ion and water channels) and carriers (transporters) are important. Generally, each class of transport proteins conducts specific molecular species in and out of the cell while preventing the passage of others, a property critical for the overall conservation of the cells internal pH and salt concentration. Both ion and water channels are highly efficient membrane pore proteins capable of transporting solutes at very high rates, up to 109 molecules per second. Carrier proteins generally have a lower turnover but are capable of transport against gradients. For both classes of proteins, their unique flux-properties make them interesting as candidates in biomimetic sensor/separation devices. An ideal sensor/separation device requires the supporting biomimetic matrix to be virtually impermeable to anything but the solute in question. In practice, however, a biomimetic support matrix will generally have finite permeabilities to water, electrolytes, and non-electrolytes. The feasibility of a biomimetic device thus depends on the relative transport contribution from both protein and biomimetic support matrix. Also the stability of the incorporated protein must be addressed and the protein-biomimetic matrix must be encapsulated in order to protect it and make it sufficiently stable in a final application. Here I will review and discuss these challenges and how they are met in some current developments of biomimetic sensor/separation devices.

Keywords: Biomaterials; Biopolymers/lipids; Biosensors; Microfluidics/microfabrication; Separations/instrumentation; Water


Large scale biomimetic membrane arrays by Jesper S. Hansen; Mark Perry; Jörg Vogel; Jesper S. Groth; Thomas Vissing; Marianne S. Larsen; Oliver Geschke; Jenny Emneús; Henrik Bohr; Claus H. Nielsen (pp. 719-727).
To establish planar biomimetic membranes across large scale partition aperture arrays, we created a disposable single-use horizontal chamber design that supports combined optical–electrical measurements. Functional lipid bilayers could easily and efficiently be established across CO2 laser micro-structured 8 × 8 aperture partition arrays with average aperture diameters of 301 ± 5 μm. We addressed the electro-physical properties of the lipid bilayers established across the micro-structured scaffold arrays by controllable reconstitution of biotechnological and physiological relevant membrane peptides and proteins. Next, we tested the scalability of the biomimetic membrane design by establishing lipid bilayers in rectangular 24 × 24 and hexagonal 24 × 27 aperture arrays, respectively. The results presented show that the design is suitable for further developments of sensitive biosensor assays, and furthermore demonstrate that the design can conveniently be scaled up to support planar lipid bilayers in large square-centimeter partition arrays. Figure Fluorescent image of a large 24 × 24 rectangular bilayer array

Keywords: Black lipid membrane; Array; Optical–electrical measurements; Membrane-spanning peptides; Membrane protein


Large scale biomimetic membrane arrays by Jesper S. Hansen; Mark Perry; Jörg Vogel; Jesper S. Groth; Thomas Vissing; Marianne S. Larsen; Oliver Geschke; Jenny Emneús; Henrik Bohr; Claus H. Nielsen (pp. 719-727).
To establish planar biomimetic membranes across large scale partition aperture arrays, we created a disposable single-use horizontal chamber design that supports combined optical–electrical measurements. Functional lipid bilayers could easily and efficiently be established across CO2 laser micro-structured 8 × 8 aperture partition arrays with average aperture diameters of 301 ± 5 μm. We addressed the electro-physical properties of the lipid bilayers established across the micro-structured scaffold arrays by controllable reconstitution of biotechnological and physiological relevant membrane peptides and proteins. Next, we tested the scalability of the biomimetic membrane design by establishing lipid bilayers in rectangular 24 × 24 and hexagonal 24 × 27 aperture arrays, respectively. The results presented show that the design is suitable for further developments of sensitive biosensor assays, and furthermore demonstrate that the design can conveniently be scaled up to support planar lipid bilayers in large square-centimeter partition arrays. Figure Fluorescent image of a large 24 × 24 rectangular bilayer array

Keywords: Black lipid membrane; Array; Optical–electrical measurements; Membrane-spanning peptides; Membrane protein


Melamine contamination by Yu-Chang Tyan; Ming-Hui Yang; Shiang-Bin Jong; Chih-Kuang Wang; Jentaie Shiea (pp. 729-735).
In the summer of 2008, serious illnesses and deaths of babies in China were linked to melamine-tainted powdered infant formula. Melamine contains several metabolites, such as ammeline, ammelide, and cyanuric acid, and has been used for the adulteration of foods or milk to increase their apparent protein content. It is assumed that melamine and its metabolites are absorbed in the gastrointestinal tract, and precipitate in the kidney to form crystals. A new tolerable daily intake of 0.2 mg kg−1 body weight was adapted by the World Health Organization in 2008. This paper reviews the variety of analytical methods that have been used for the analysis of melamine in food. The limit of detection of these various methods is 0.05–100 ppm. The maximum acceptable concentration in food has been set at 50 ppb by the US FDA. A fast and ultrasensitive procedure for screening, detection, and characterization of melamine and its derivative compounds needs to be established. Currently, mass-spectrometry technologies provide an alternative to derivatization for regulatory analysis of food.

Keywords: Melamine; Cyanuric acid; High-performance liquid chromatography-tandem mass spectrometry; Gas chromatography-tandem mass spectrometry; Isotope dilution


Melamine contamination by Yu-Chang Tyan; Ming-Hui Yang; Shiang-Bin Jong; Chih-Kuang Wang; Jentaie Shiea (pp. 729-735).
In the summer of 2008, serious illnesses and deaths of babies in China were linked to melamine-tainted powdered infant formula. Melamine contains several metabolites, such as ammeline, ammelide, and cyanuric acid, and has been used for the adulteration of foods or milk to increase their apparent protein content. It is assumed that melamine and its metabolites are absorbed in the gastrointestinal tract, and precipitate in the kidney to form crystals. A new tolerable daily intake of 0.2 mg kg−1 body weight was adapted by the World Health Organization in 2008. This paper reviews the variety of analytical methods that have been used for the analysis of melamine in food. The limit of detection of these various methods is 0.05–100 ppm. The maximum acceptable concentration in food has been set at 50 ppb by the US FDA. A fast and ultrasensitive procedure for screening, detection, and characterization of melamine and its derivative compounds needs to be established. Currently, mass-spectrometry technologies provide an alternative to derivatization for regulatory analysis of food.

Keywords: Melamine; Cyanuric acid; High-performance liquid chromatography-tandem mass spectrometry; Gas chromatography-tandem mass spectrometry; Isotope dilution


Surfactant-coated carbon nanotubes for the liquid–liquid extraction of phthalates and other migrants in virgin olive oils by Silvia López-Feria; Rafael Lucena; Soledad Cárdenas; Miguel Valcárcel (pp. 737-746).
This article presents a novel application of the surfactant-coated carbon nanotubes for the joint extraction of phthalates, xylene isomers and styrene from virgin olive oils. For this purpose, two carbon nanotubes, multi-walled and single-walled carbon nanotubes, were evaluated, the former providing the better results. The target migrants were liquid–liquid extracted from the virgin olive oil samples by means of the carbon nanotube pseudophase, and the aqueous extracts were analysed by headspace-gas chromatography-mass spectrometry. Sodium chloride was added to favour the release of the analytes to the gaseous phase of the vial. The comparison of the chromatograms obtained with the direct analysis reveals a sensitivity enhancement with the use of the surfactant-coated carbon nanotubes, especially for the phthalates, which can be ascribed to the π–π interaction between the aromatic ring and the nanotube surface. Limits of detection were between 6 and 30 µgL−1 for 1 mL of sample. The precision of the method was better than 7.6% and the recoveries higher than 92%. Finally, the influence of the package material and the storage conditions on the concentration of the analytes was accomplished, and it was found that the concentration of phthalates increases with time in all the packages studied, while the more volatile compounds were found to decrease under the same conditions.

Keywords: Phthalates; Xylene isomers; Styrene; Liquid–liquid extraction; Carbon nanotubes; Headspace-gas chromatography-mass spectrometry


Surfactant-coated carbon nanotubes for the liquid–liquid extraction of phthalates and other migrants in virgin olive oils by Silvia López-Feria; Rafael Lucena; Soledad Cárdenas; Miguel Valcárcel (pp. 737-746).
This article presents a novel application of the surfactant-coated carbon nanotubes for the joint extraction of phthalates, xylene isomers and styrene from virgin olive oils. For this purpose, two carbon nanotubes, multi-walled and single-walled carbon nanotubes, were evaluated, the former providing the better results. The target migrants were liquid–liquid extracted from the virgin olive oil samples by means of the carbon nanotube pseudophase, and the aqueous extracts were analysed by headspace-gas chromatography-mass spectrometry. Sodium chloride was added to favour the release of the analytes to the gaseous phase of the vial. The comparison of the chromatograms obtained with the direct analysis reveals a sensitivity enhancement with the use of the surfactant-coated carbon nanotubes, especially for the phthalates, which can be ascribed to the π–π interaction between the aromatic ring and the nanotube surface. Limits of detection were between 6 and 30 µgL−1 for 1 mL of sample. The precision of the method was better than 7.6% and the recoveries higher than 92%. Finally, the influence of the package material and the storage conditions on the concentration of the analytes was accomplished, and it was found that the concentration of phthalates increases with time in all the packages studied, while the more volatile compounds were found to decrease under the same conditions.

Keywords: Phthalates; Xylene isomers; Styrene; Liquid–liquid extraction; Carbon nanotubes; Headspace-gas chromatography-mass spectrometry


Magnetic track array for efficient bead capture in microchannels by Mélanie Abonnenc; Anne-Laure Gassner; Jacques Morandini; Jacques Josserand; Hubert H. Girault (pp. 747-757).
Magnetism-based microsystems, as those dedicated to immunoaffinity separations or (bio)chemical reactions, take benefit of the large surface area-to-volume ratio provided by the immobilized magnetic beads, thus increasing the sensitivity of the analysis. As the sensitivity is directly linked to the efficiency of the magnetic bead capture, this paper presents a simple method to enhance the capture in a microchannel. Considering a microchannel surrounded by two rectangular permanent magnets of different length (L m = 2, 5, 10 mm) placed in attraction, it is shown that the amount of trapped beads is limited by the magnetic forces mainly located at the magnet edges. To overcome this limitation, a polyethylene terephthalate (PET) microchip with an integrated magnetic track array has been prototyped by laser photo-ablation. The magnetic force is therefore distributed all along the magnet length. It results in a multi-plug bead capture, observed by microscope imaging, with a magnetic force value locally enhanced. The relative amount of beads, and so the specific binding surface for further immunoassays, presents a significant increase of 300% for the largest magnets. The influence of the track geometry and relative permeability on the magnetic force was studied by numerical simulations, for the microchip operating with 2-mm-long magnets.

Keywords: Microfluidics; Magnetism; Permanent magnet; Magnetic bead; Ink; Magnetic track array; Photo-ablation; Polymer; Numerical simulation


Magnetic track array for efficient bead capture in microchannels by Mélanie Abonnenc; Anne-Laure Gassner; Jacques Morandini; Jacques Josserand; Hubert H. Girault (pp. 747-757).
Magnetism-based microsystems, as those dedicated to immunoaffinity separations or (bio)chemical reactions, take benefit of the large surface area-to-volume ratio provided by the immobilized magnetic beads, thus increasing the sensitivity of the analysis. As the sensitivity is directly linked to the efficiency of the magnetic bead capture, this paper presents a simple method to enhance the capture in a microchannel. Considering a microchannel surrounded by two rectangular permanent magnets of different length (L m = 2, 5, 10 mm) placed in attraction, it is shown that the amount of trapped beads is limited by the magnetic forces mainly located at the magnet edges. To overcome this limitation, a polyethylene terephthalate (PET) microchip with an integrated magnetic track array has been prototyped by laser photo-ablation. The magnetic force is therefore distributed all along the magnet length. It results in a multi-plug bead capture, observed by microscope imaging, with a magnetic force value locally enhanced. The relative amount of beads, and so the specific binding surface for further immunoassays, presents a significant increase of 300% for the largest magnets. The influence of the track geometry and relative permeability on the magnetic force was studied by numerical simulations, for the microchip operating with 2-mm-long magnets.

Keywords: Microfluidics; Magnetism; Permanent magnet; Magnetic bead; Ink; Magnetic track array; Photo-ablation; Polymer; Numerical simulation


Direct immobilization of functional single-chain variable fragment antibodies (scFvs) onto a polystyrene plate by genetic fusion of a polystyrene-binding peptide (PS-tag) by Yoichi Kumada; Kyoko Hamasaki; Yuki Shiritani; Aya Nakagawa; Daisuke Kuroki; Takuhito Ohse; Dong Hwan Choi; Yoshio Katakura; Michimasa Kishimoto (pp. 759-765).
Single-chain Fv antibodies (scFv) genetically fused with polystyrene-binding peptides (PS-tags, (PS19-1; RAFIASRRIRRP, PS19-6; RIIIRRIRR)) were generated by recombinant Escherichia coli for direct and site-specific immobilization of scFv on polystyrene supports with high antigen-binding activity. PS-tag-fused scFvs (scFv-PS-tags) specific for human C-reactive protein (CRP) were successfully over-expressed as an inclusion body and were refolded using the batch-dilution method. When scFv-PS-tags were immobilized on a hydrophilic PS (phi-PS) plate in the presence of Tween 20, they showed high antigen-binding activity comparable to, or greater than, that of a whole monoclonal antibody (mAb) on a hydrophobic PS (pho-PS) plate, which has been the exclusive method for enzyme-linked immunosorbent assay (ELISA). Furthermore, when a scFv-PS-tag was used as a ligand antibody in one- and two-step ELISA, the assay time was reduced without loss of sensitivity. These results indicate that strong and specific attachment of PS-tags onto the phi-PS surface prevented scFv conformational changes and consequently, the high antigen-binding activities of scFvs were preserved. Nearly identical results were obtained by use of PS-tag-fused scFvs with different VH/VL pairs. Therefore, a variety of scFvs could be functionalized onto phi-PS plates by genetic fusion of PS-tags. ScFv-PS-tags, which possess high antigen-binding activity on the phi-PS plate, are more useful ligand antibodies than whole mAbs. Thus, scFv-PS-tags are applicable in both clinical diagnosis and proteomic research.

Keywords: Site-specific immobilization; Orientation control; Single-chain fragment of variable domain (scFv); Polystyrene-binding peptide (PS-tag); Polystyrene; One-step ELISA


Direct immobilization of functional single-chain variable fragment antibodies (scFvs) onto a polystyrene plate by genetic fusion of a polystyrene-binding peptide (PS-tag) by Yoichi Kumada; Kyoko Hamasaki; Yuki Shiritani; Aya Nakagawa; Daisuke Kuroki; Takuhito Ohse; Dong Hwan Choi; Yoshio Katakura; Michimasa Kishimoto (pp. 759-765).
Single-chain Fv antibodies (scFv) genetically fused with polystyrene-binding peptides (PS-tags, (PS19-1; RAFIASRRIRRP, PS19-6; RIIIRRIRR)) were generated by recombinant Escherichia coli for direct and site-specific immobilization of scFv on polystyrene supports with high antigen-binding activity. PS-tag-fused scFvs (scFv-PS-tags) specific for human C-reactive protein (CRP) were successfully over-expressed as an inclusion body and were refolded using the batch-dilution method. When scFv-PS-tags were immobilized on a hydrophilic PS (phi-PS) plate in the presence of Tween 20, they showed high antigen-binding activity comparable to, or greater than, that of a whole monoclonal antibody (mAb) on a hydrophobic PS (pho-PS) plate, which has been the exclusive method for enzyme-linked immunosorbent assay (ELISA). Furthermore, when a scFv-PS-tag was used as a ligand antibody in one- and two-step ELISA, the assay time was reduced without loss of sensitivity. These results indicate that strong and specific attachment of PS-tags onto the phi-PS surface prevented scFv conformational changes and consequently, the high antigen-binding activities of scFvs were preserved. Nearly identical results were obtained by use of PS-tag-fused scFvs with different VH/VL pairs. Therefore, a variety of scFvs could be functionalized onto phi-PS plates by genetic fusion of PS-tags. ScFv-PS-tags, which possess high antigen-binding activity on the phi-PS plate, are more useful ligand antibodies than whole mAbs. Thus, scFv-PS-tags are applicable in both clinical diagnosis and proteomic research.

Keywords: Site-specific immobilization; Orientation control; Single-chain fragment of variable domain (scFv); Polystyrene-binding peptide (PS-tag); Polystyrene; One-step ELISA


Electrochemical monitoring of binding between wheat germ agglutinin and cellohexose-modified magnetic microbeads by Kazuharu Sugawara; Asako Yugami; Hideki Kuramitz (pp. 767-772).
An electrochemical method that uses glucose labeled with an electroactive compound was developed to evaluate the binding of wheat germ agglutinin (WGA) to cellohexose-modified magnetic microbeads. Cellohexose was attached to amino groups on the magnetic bead surface via formation of a Schiff's base. The labeled glucose acts as an electrochemical probe to monitor binding events between WGA and the cellohexose-modified beads. For a known quantity of cellohexose-modified beads, binding of WGA with cellohexose-modified beads was evaluated based on changes in electrochemical response of the labeled glucose. In particular, the peak current decreased as the concentration of WGA increased. Furthermore, the binding affinities of WGA for beads modified with four different cello-oligosaccharides were systematically compared using a voltammetric method. Figure We captured wheat germ agglutinin on cellohexose-modified magnetic microbeads and monitored the binding with an electrochemical procedure

Keywords: Wheat germ agglutinin; Cellohexose; Electrochemical probe; Magnetic microbead


Electrochemical monitoring of binding between wheat germ agglutinin and cellohexose-modified magnetic microbeads by Kazuharu Sugawara; Asako Yugami; Hideki Kuramitz (pp. 767-772).
An electrochemical method that uses glucose labeled with an electroactive compound was developed to evaluate the binding of wheat germ agglutinin (WGA) to cellohexose-modified magnetic microbeads. Cellohexose was attached to amino groups on the magnetic bead surface via formation of a Schiff's base. The labeled glucose acts as an electrochemical probe to monitor binding events between WGA and the cellohexose-modified beads. For a known quantity of cellohexose-modified beads, binding of WGA with cellohexose-modified beads was evaluated based on changes in electrochemical response of the labeled glucose. In particular, the peak current decreased as the concentration of WGA increased. Furthermore, the binding affinities of WGA for beads modified with four different cello-oligosaccharides were systematically compared using a voltammetric method. Figure We captured wheat germ agglutinin on cellohexose-modified magnetic microbeads and monitored the binding with an electrochemical procedure

Keywords: Wheat germ agglutinin; Cellohexose; Electrochemical probe; Magnetic microbead


RNAi and iTRAQ reagents united: targeted quantitation of siRNA-mediated protein silencing in human cells by A. Abdrakhmanova; R. Schlichting; C. L. Hunter; M. Glueckmann; C. Lenz; C. J. Echeverri; B. Soennichsen; A. Jung; C. Weiss-Haljiti (pp. 773-785).
Bridging the gap between functional genomics and traditional molecular cell biology is a challenge of the next decade. Here, we are aiming to find routines for targeted quantitation of protein silencing in response to RNAi based on complex cellular lysates. A workflow was established adapting siRNA treatment, processing the sample, generating isobaric iTRAQ®-reagent-labeled peptides, and analyzing the sample applying MRM-based peptide quantitation to verify protein silencing on a 4000 QTRAP LC/MS/MS mass spectrometer. Subsequently, eight targets were analyzed, mostly with two siRNA designs. Although transcript and protein silencing correlated, the downregulation on the protein level was less pronounced. A time-course analysis of the chaperon HSPA9/mortalin indicated a delayed kinetic of protein versus transcript silencing. Further, the analysis of the functional response on the example of HSD17B4, a multifunctional enzyme essential to generate precursors for cholesterol biosynthesis, confirmed that strong silencing on the transcript level accompanied by moderate reduction of protein is sufficient to generate a physiological significant response. Fifty percent protein silencing resulted in a 3.5-fold induction of low-density lipoprotein and therefore cholesterol uptake in human liver cells. The established routines pave the way for the development of targeted protein quantitation assays suitable for target and biomarker validation.

Keywords: RNAi; Protein silencing; Quantitative proteomics; iTRAQ labeling; Multiple-reaction monitoring; Huh7 liver cells


RNAi and iTRAQ reagents united: targeted quantitation of siRNA-mediated protein silencing in human cells by A. Abdrakhmanova; R. Schlichting; C. L. Hunter; M. Glueckmann; C. Lenz; C. J. Echeverri; B. Soennichsen; A. Jung; C. Weiss-Haljiti (pp. 773-785).
Bridging the gap between functional genomics and traditional molecular cell biology is a challenge of the next decade. Here, we are aiming to find routines for targeted quantitation of protein silencing in response to RNAi based on complex cellular lysates. A workflow was established adapting siRNA treatment, processing the sample, generating isobaric iTRAQ®-reagent-labeled peptides, and analyzing the sample applying MRM-based peptide quantitation to verify protein silencing on a 4000 QTRAP LC/MS/MS mass spectrometer. Subsequently, eight targets were analyzed, mostly with two siRNA designs. Although transcript and protein silencing correlated, the downregulation on the protein level was less pronounced. A time-course analysis of the chaperon HSPA9/mortalin indicated a delayed kinetic of protein versus transcript silencing. Further, the analysis of the functional response on the example of HSD17B4, a multifunctional enzyme essential to generate precursors for cholesterol biosynthesis, confirmed that strong silencing on the transcript level accompanied by moderate reduction of protein is sufficient to generate a physiological significant response. Fifty percent protein silencing resulted in a 3.5-fold induction of low-density lipoprotein and therefore cholesterol uptake in human liver cells. The established routines pave the way for the development of targeted protein quantitation assays suitable for target and biomarker validation.

Keywords: RNAi; Protein silencing; Quantitative proteomics; iTRAQ labeling; Multiple-reaction monitoring; Huh7 liver cells


Simultaneous cell capture and induction of apoptosis using an anti-CD95 affinity microdevice by Randall D. Reif; Michelle M. Martinez; Kelong Wang; Dimitri Pappas (pp. 787-795).
A microfluidic device is designed and demonstrated for the simultaneous capture and induction of apoptosis in Jurkat cells. In this unique case, the cell capture event initiates a biological process. The device features a single channel made from poly(dimethylsiloxane) sealed to a glass substrate. The channel is coated with a series of reagents used in affinity chromatography separations of cells. In this case, the antibody used to capture the cells is functional anti-CD95 which captures the cells by binding to the Fas receptor on the cell membrane and, at the same time, inducing apoptosis via the caspase 8 pathway. Cells retained on the surface of the channel are known to be induced to undergo apoptosis. Medium is flowed slowly through the channel to maintain cell viability while the cells undergo apoptosis. After 3 h, staining with Annexin V-PE and 7-AAD revealed that 43.5% of the cells bound to the anti-CD95 coated channel are apoptotic, whereas 7.9% of cultured Jurkat cells induced with anti-CD95 for 3 h and stained in the same way were determined to be apoptotic by flow cytometry. The device provides a method of determining when apoptosis is induced, maintaining cell viability for long-term analysis and observing cells in real time as they are exposed to reagents that affect apoptosis. In the future, the device will be an invaluable tool for the study of the temporal dynamics of apoptosis.

Keywords: Apoptosis; Affinity separation; Caspase inhibition; Light microscopy


Simultaneous cell capture and induction of apoptosis using an anti-CD95 affinity microdevice by Randall D. Reif; Michelle M. Martinez; Kelong Wang; Dimitri Pappas (pp. 787-795).
A microfluidic device is designed and demonstrated for the simultaneous capture and induction of apoptosis in Jurkat cells. In this unique case, the cell capture event initiates a biological process. The device features a single channel made from poly(dimethylsiloxane) sealed to a glass substrate. The channel is coated with a series of reagents used in affinity chromatography separations of cells. In this case, the antibody used to capture the cells is functional anti-CD95 which captures the cells by binding to the Fas receptor on the cell membrane and, at the same time, inducing apoptosis via the caspase 8 pathway. Cells retained on the surface of the channel are known to be induced to undergo apoptosis. Medium is flowed slowly through the channel to maintain cell viability while the cells undergo apoptosis. After 3 h, staining with Annexin V-PE and 7-AAD revealed that 43.5% of the cells bound to the anti-CD95 coated channel are apoptotic, whereas 7.9% of cultured Jurkat cells induced with anti-CD95 for 3 h and stained in the same way were determined to be apoptotic by flow cytometry. The device provides a method of determining when apoptosis is induced, maintaining cell viability for long-term analysis and observing cells in real time as they are exposed to reagents that affect apoptosis. In the future, the device will be an invaluable tool for the study of the temporal dynamics of apoptosis.

Keywords: Apoptosis; Affinity separation; Caspase inhibition; Light microscopy


Automated microextraction sample preparation coupled on-line to FT-ICR-MS: application to desalting and concentration of river and marine dissolved organic matter by Gabriel Morales-Cid; Istvan Gebefugi; Basem Kanawati; Mourad Harir; Norbert Hertkorn; Ramón Rosselló-Mora; Philippe Schmitt-Kopplin (pp. 797-807).
Sample preparation procedures are in most cases sample- and time-consuming and commonly require the use of a large amount of solvents. Automation in this regard can optimize the minimal-needed injection volume and the solvent consumption will be efficiently reduced. A new fully automated sample desalting and pre-concentration technique employing microextraction by packed sorbents (MEPS) cartridges is implemented and coupled to an ion cyclotron resonance Fourier-transform mass spectrometer (ICR-FT/MS). The performance of non-target mass spectrometric analysis is compared for the automated versus off-line sample preparation for several samples of aqueous natural organic matter. This approach can be generalized for any metabolite profiling or metabolome analysis of biological materials but was optimized herein using a well characterized but highly complex organic mixture: a surface water and its well-characterized natural organic matter and a marine sample having a highly salt charge and enabling to validate the presented automatic system for salty samples. The analysis of Suwannee River water showed selective C18-MEPS enrichment of chemical signatures with average H/C and O/C elemental ratios and loss of both highly polar and highly aromatic structures from the original sample. Automated on-line application to marine samples showed desalting and different chemical signatures from surface to bottom water. Relative comparison of structural footprints with the C18-concentration/desalting procedure however enabled to demonstrate that the surface water film was more concentrated in surface-active components of natural (fatty acids) and anthropogenic origin (sulfur-containing surfactants). Overall, the relative standard deviation distribution in terms of peak intensity was improved by automating the proposed on-line method. Figure The efficient use of MEPS technique in automated form with high resolution FT-ICR-MS gives a selective spectrum, which reveals rich see water organic content. The shown high sensitivity of detected organic matter is due to effective desalting achieved by automated MEPS coupling to FT-ICR-MS technique.

Keywords: ICR-FT/MS; MEPS; Suwannee river; Marine water samples; On-line water analysis


Automated microextraction sample preparation coupled on-line to FT-ICR-MS: application to desalting and concentration of river and marine dissolved organic matter by Gabriel Morales-Cid; Istvan Gebefugi; Basem Kanawati; Mourad Harir; Norbert Hertkorn; Ramón Rosselló-Mora; Philippe Schmitt-Kopplin (pp. 797-807).
Sample preparation procedures are in most cases sample- and time-consuming and commonly require the use of a large amount of solvents. Automation in this regard can optimize the minimal-needed injection volume and the solvent consumption will be efficiently reduced. A new fully automated sample desalting and pre-concentration technique employing microextraction by packed sorbents (MEPS) cartridges is implemented and coupled to an ion cyclotron resonance Fourier-transform mass spectrometer (ICR-FT/MS). The performance of non-target mass spectrometric analysis is compared for the automated versus off-line sample preparation for several samples of aqueous natural organic matter. This approach can be generalized for any metabolite profiling or metabolome analysis of biological materials but was optimized herein using a well characterized but highly complex organic mixture: a surface water and its well-characterized natural organic matter and a marine sample having a highly salt charge and enabling to validate the presented automatic system for salty samples. The analysis of Suwannee River water showed selective C18-MEPS enrichment of chemical signatures with average H/C and O/C elemental ratios and loss of both highly polar and highly aromatic structures from the original sample. Automated on-line application to marine samples showed desalting and different chemical signatures from surface to bottom water. Relative comparison of structural footprints with the C18-concentration/desalting procedure however enabled to demonstrate that the surface water film was more concentrated in surface-active components of natural (fatty acids) and anthropogenic origin (sulfur-containing surfactants). Overall, the relative standard deviation distribution in terms of peak intensity was improved by automating the proposed on-line method. Figure The efficient use of MEPS technique in automated form with high resolution FT-ICR-MS gives a selective spectrum, which reveals rich see water organic content. The shown high sensitivity of detected organic matter is due to effective desalting achieved by automated MEPS coupling to FT-ICR-MS technique.

Keywords: ICR-FT/MS; MEPS; Suwannee river; Marine water samples; On-line water analysis


Identification and quantification of glutathione and phytochelatins from Chlorella vulgaris by RP-HPLC ESI-MS/MS and oxygen-free extraction by Denina B. D. Simmons; Allison R. Hayward; Thomas C. Hutchinson; R. J. Neil Emery (pp. 809-817).
Phytochelatins are short, cysteine-containing, detoxification peptides produced by plants, algae, and fungi in response to heavy metal exposure. These peptides auto-oxidize easily. Current extraction protocols do not adequately address losses of phytochelatins because of their oxidation and the use of indirect methods for quantification. Method enhancements include the use of an argon environment during extraction to reduce auto-oxidation, the use of glycine-13C2-labeled glutathione as an internal standard, and an electrospray ionization source with a triple quadrupole mass spectrometer as a detector. The method-detection limits were 0.081 μM for glutathione, 0.440 μM for phytochelatin 2, and 0.120 μM for phytochelatin 3. These detection limits were comparable to similar studies and were not compromised incorporating these adjustments. The use of a labeled internal standard and an inert gaseous environment during sample preparation greatly improved calibration linearity and sensitivity. Furthermore, phytochelatin degradation was significantly reduced and more accurately tracked. Previous studies involving phytochelatin analyses have likely been subject to higher variability caused by this propensity for phytochelatins to degrade rapidly in air. The method adjustments were simple and cost-effective and allowed phytochelatin analyses to be performed for hours at a time with minimal auto-oxidation. Figure This figure was created using Adobe Photoshop CS3 (San Jose, CA). It displays an image of a chromatogram from our manuscript (a phytochelatin extraction) overlaid upon an image of Chlorella vulgaris and laboratory equipment and materials used in our methods.

Keywords: Phytochelatins; Chlorella vulgaris ; Auto-oxidation; HPLC-MS; Internal standard; ESI-MS/MS


Identification and quantification of glutathione and phytochelatins from Chlorella vulgaris by RP-HPLC ESI-MS/MS and oxygen-free extraction by Denina B. D. Simmons; Allison R. Hayward; Thomas C. Hutchinson; R. J. Neil Emery (pp. 809-817).
Phytochelatins are short, cysteine-containing, detoxification peptides produced by plants, algae, and fungi in response to heavy metal exposure. These peptides auto-oxidize easily. Current extraction protocols do not adequately address losses of phytochelatins because of their oxidation and the use of indirect methods for quantification. Method enhancements include the use of an argon environment during extraction to reduce auto-oxidation, the use of glycine-13C2-labeled glutathione as an internal standard, and an electrospray ionization source with a triple quadrupole mass spectrometer as a detector. The method-detection limits were 0.081 μM for glutathione, 0.440 μM for phytochelatin 2, and 0.120 μM for phytochelatin 3. These detection limits were comparable to similar studies and were not compromised incorporating these adjustments. The use of a labeled internal standard and an inert gaseous environment during sample preparation greatly improved calibration linearity and sensitivity. Furthermore, phytochelatin degradation was significantly reduced and more accurately tracked. Previous studies involving phytochelatin analyses have likely been subject to higher variability caused by this propensity for phytochelatins to degrade rapidly in air. The method adjustments were simple and cost-effective and allowed phytochelatin analyses to be performed for hours at a time with minimal auto-oxidation. Figure This figure was created using Adobe Photoshop CS3 (San Jose, CA). It displays an image of a chromatogram from our manuscript (a phytochelatin extraction) overlaid upon an image of Chlorella vulgaris and laboratory equipment and materials used in our methods.

Keywords: Phytochelatins; Chlorella vulgaris ; Auto-oxidation; HPLC-MS; Internal standard; ESI-MS/MS


Analysis of drugs of abuse in wastewater by hydrophilic interaction liquid chromatography–tandem mass spectrometry by Alexander L. N. van Nuijs; Isabela Tarcomnicu; Lieven Bervoets; Ronny Blust; Philippe G. Jorens; Hugo Neels; Adrian Covaci (pp. 819-828).
The simultaneous analysis of nine drugs of abuse (DOAs) and their metabolites (amphetamine, methamphetamine, methylenedioxymethamphetamine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, cocaine, benzoylecgonine, ecgonine methyl ester and 6-monoacetylmorphine) in wastewater based on hydrophilic interaction liquid chromatography (HILIC) coupled to tandem mass spectrometry (MS/MS) was optimised and validated. For each analyte, the deuterated analogue was used for quantification. The separation by HILIC showed good performance for all compounds, especially for the hydrophilic compounds, which elute early (amphetamine-like stimulants) or show no retention (ecgonine methyl ester) in reversed-phase liquid chromatography. Sample preparation based on solid-phase extraction was optimised by comparing Oasis HLB and Oasis MCX sorbents for various parameters such as sample pH, amount of sorbent bed and washing solvent. The method was validated for each compound by assessing the following parameters (following International Conference on Harmonisation guidelines): specificity, limit of quantification (LOQ), linearity, accuracy, precision, recovery and matrix effects. LOQs were 2 ng/L for 6-monoacetylmorphine, ecgonine methyl ester and amphetamine and 1 ng/L for the rest of the compounds, corresponding with the lowest point in the calibration curve. Except for 6-monoacetylmorphine, all compounds were detected from 1 to 819 ng/L in influent wastewater samples (n = 12) collected from 11 different wastewater treatment plants across Belgium. The presence of ecgonine methyl ester in wastewater could be demonstrated for the first time. In the future, the new HILIC–MS/MS method will be applied to assess the use of DOAs in Belgium using the “sewage epidemiology” approach.

Keywords: Drugs of abuse; Metabolites; HILIC–MS/MS; Wastewater; SPE; Validation


Analysis of drugs of abuse in wastewater by hydrophilic interaction liquid chromatography–tandem mass spectrometry by Alexander L. N. van Nuijs; Isabela Tarcomnicu; Lieven Bervoets; Ronny Blust; Philippe G. Jorens; Hugo Neels; Adrian Covaci (pp. 819-828).
The simultaneous analysis of nine drugs of abuse (DOAs) and their metabolites (amphetamine, methamphetamine, methylenedioxymethamphetamine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, cocaine, benzoylecgonine, ecgonine methyl ester and 6-monoacetylmorphine) in wastewater based on hydrophilic interaction liquid chromatography (HILIC) coupled to tandem mass spectrometry (MS/MS) was optimised and validated. For each analyte, the deuterated analogue was used for quantification. The separation by HILIC showed good performance for all compounds, especially for the hydrophilic compounds, which elute early (amphetamine-like stimulants) or show no retention (ecgonine methyl ester) in reversed-phase liquid chromatography. Sample preparation based on solid-phase extraction was optimised by comparing Oasis HLB and Oasis MCX sorbents for various parameters such as sample pH, amount of sorbent bed and washing solvent. The method was validated for each compound by assessing the following parameters (following International Conference on Harmonisation guidelines): specificity, limit of quantification (LOQ), linearity, accuracy, precision, recovery and matrix effects. LOQs were 2 ng/L for 6-monoacetylmorphine, ecgonine methyl ester and amphetamine and 1 ng/L for the rest of the compounds, corresponding with the lowest point in the calibration curve. Except for 6-monoacetylmorphine, all compounds were detected from 1 to 819 ng/L in influent wastewater samples (n = 12) collected from 11 different wastewater treatment plants across Belgium. The presence of ecgonine methyl ester in wastewater could be demonstrated for the first time. In the future, the new HILIC–MS/MS method will be applied to assess the use of DOAs in Belgium using the “sewage epidemiology” approach.

Keywords: Drugs of abuse; Metabolites; HILIC–MS/MS; Wastewater; SPE; Validation


Collagen types analysis and differentiation by FTIR spectroscopy by Karima Belbachir; Razia Noreen; Gilles Gouspillou; Cyril Petibois (pp. 829-837).
Abnormal formation and organization of collagen network is commonly observed in many organ pathologies, but analytical techniques able to reveal the collagen biodistribution are still lacking. In this study, Fourier-transform infrared (FTIR) spectroscopy has been used to analyze type I, III, IV, V, and VI collagens, the most important compounds of connective tissues. A robust classification of 30 FTIR spectra per collagen type could be obtained by using a combination of four spectral intervals [ν(C=O) absorption of amide I (1,700–1,600 cm−1), δ(CH2), and δ(CH3) absorptions (1,480–1,350 cm−1), ν(C–N), and δ(N–H) absorptions of amide III (1,300–1,180 cm−1), and ν(C–O) and ν(C–O–C) absorptions of carbohydrate moieties (1,100–1,005 cm−1)]. Then, a submolecular justification of this classification model was sought using a curve fitting analysis of the four spectral intervals. Results demonstrated that every spectral interval used for the classification contained highly discriminant absorption bands between all collagen types (multivariate analysis of variance, p < 0.01; Dunnett's T3 post hoc test, p < 0.05). All conditions seem thus joined to make FTIR spectroscopy and imaging major tools for implementing innovative methods in the field of molecular histology, which would be very helpful for the diagnosis of a wide range of pathologies.

Keywords: FTIR spectroscopy; Collagens; Connective tissue; Molecular structure; Classification


Collagen types analysis and differentiation by FTIR spectroscopy by Karima Belbachir; Razia Noreen; Gilles Gouspillou; Cyril Petibois (pp. 829-837).
Abnormal formation and organization of collagen network is commonly observed in many organ pathologies, but analytical techniques able to reveal the collagen biodistribution are still lacking. In this study, Fourier-transform infrared (FTIR) spectroscopy has been used to analyze type I, III, IV, V, and VI collagens, the most important compounds of connective tissues. A robust classification of 30 FTIR spectra per collagen type could be obtained by using a combination of four spectral intervals [ν(C=O) absorption of amide I (1,700–1,600 cm−1), δ(CH2), and δ(CH3) absorptions (1,480–1,350 cm−1), ν(C–N), and δ(N–H) absorptions of amide III (1,300–1,180 cm−1), and ν(C–O) and ν(C–O–C) absorptions of carbohydrate moieties (1,100–1,005 cm−1)]. Then, a submolecular justification of this classification model was sought using a curve fitting analysis of the four spectral intervals. Results demonstrated that every spectral interval used for the classification contained highly discriminant absorption bands between all collagen types (multivariate analysis of variance, p < 0.01; Dunnett's T3 post hoc test, p < 0.05). All conditions seem thus joined to make FTIR spectroscopy and imaging major tools for implementing innovative methods in the field of molecular histology, which would be very helpful for the diagnosis of a wide range of pathologies.

Keywords: FTIR spectroscopy; Collagens; Connective tissue; Molecular structure; Classification


Analytical control of adjuvants in herbicide formulations by NIR spectroscopy by Marcelo Blanco; David Zamora; Joaquim Planells; Rufí Mulero (pp. 839-844).
Herbicide preparations, which typically consist of an active compound and an adjuvant, are formulated in such a way as to facilitate rapid dispersion of the pesticide and its transfer across the fat cuticle of plant leaves. The adjuvant usually comprises various substances including an oil, water, and one or more emulsifiers for increased stability. The difficulty of analyzing these mixtures has severely restricted the ability to control herbicide preparations, which must usually be characterized in terms of global, non-specific parameters such as the acid, saponification, and hydroxyl values. Near-infrared spectroscopy is an expeditious analytical technique requiring no sample preparation and affording multi-determinations in complex mixtures. In this work, we used laboratory-made samples to construct PLS multivariate models for determining the components of the adjuvant mixture in a herbicide preparation. The best ensuing model was validated and used to analyze industrial preparations with good results.

Keywords: Emulsions; Surfactants; Near-infrared spectroscopy; Multivariate calibration


Analytical control of adjuvants in herbicide formulations by NIR spectroscopy by Marcelo Blanco; David Zamora; Joaquim Planells; Rufí Mulero (pp. 839-844).
Herbicide preparations, which typically consist of an active compound and an adjuvant, are formulated in such a way as to facilitate rapid dispersion of the pesticide and its transfer across the fat cuticle of plant leaves. The adjuvant usually comprises various substances including an oil, water, and one or more emulsifiers for increased stability. The difficulty of analyzing these mixtures has severely restricted the ability to control herbicide preparations, which must usually be characterized in terms of global, non-specific parameters such as the acid, saponification, and hydroxyl values. Near-infrared spectroscopy is an expeditious analytical technique requiring no sample preparation and affording multi-determinations in complex mixtures. In this work, we used laboratory-made samples to construct PLS multivariate models for determining the components of the adjuvant mixture in a herbicide preparation. The best ensuing model was validated and used to analyze industrial preparations with good results.

Keywords: Emulsions; Surfactants; Near-infrared spectroscopy; Multivariate calibration


High-performance liquid chromatography purification of biosurfactant isoforms produced by a marine bacterium by C. Sivapathasekaran; Soumen Mukherjee; Ramapati Samanta; Ramkrishna Sen (pp. 845-854).
A marine Bacillus strain produced biosurfactant during its growth in a defined glucose-containing medium. An efficient method for separation and purification of biosurfactant isoforms was developed and optimized in high-performance liquid chromatography (HPLC) by manipulating solvent gradient program and flow rates. Starting with an initial run time of 60 min, the final optimized method had a significantly reduced run time of 20 min. By using this method, all the surface-active isoforms (fractions A–D) were eluted within 12 min of elution with much shortened retention time of each component. The purity levels of the isoforms were enhanced using the optimized method as evident from their lower CMC values. Among the four surface-active fractions, antimicrobial action was solely displayed by HPLC fraction A. FTIR analysis revealed all the HPLC fractions to be lipopeptide in nature and MALDI-ToF mass spectral analysis showed that these belonged to the fengycin family containing C15, C16, and C17 fengycins.

Keywords: Lipopeptide; HPLC; CMC; FTIR; MALDI-ToF; Fengycins; Antimicrobial activity


High-performance liquid chromatography purification of biosurfactant isoforms produced by a marine bacterium by C. Sivapathasekaran; Soumen Mukherjee; Ramapati Samanta; Ramkrishna Sen (pp. 845-854).
A marine Bacillus strain produced biosurfactant during its growth in a defined glucose-containing medium. An efficient method for separation and purification of biosurfactant isoforms was developed and optimized in high-performance liquid chromatography (HPLC) by manipulating solvent gradient program and flow rates. Starting with an initial run time of 60 min, the final optimized method had a significantly reduced run time of 20 min. By using this method, all the surface-active isoforms (fractions A–D) were eluted within 12 min of elution with much shortened retention time of each component. The purity levels of the isoforms were enhanced using the optimized method as evident from their lower CMC values. Among the four surface-active fractions, antimicrobial action was solely displayed by HPLC fraction A. FTIR analysis revealed all the HPLC fractions to be lipopeptide in nature and MALDI-ToF mass spectral analysis showed that these belonged to the fengycin family containing C15, C16, and C17 fengycins.

Keywords: Lipopeptide; HPLC; CMC; FTIR; MALDI-ToF; Fengycins; Antimicrobial activity


A highly selective polymeric membrane barium(II) electrode based on a macrocyclic tetrabasic ligand as neutral carrier by Yu-Hua Ma; Ruo Yuan; Ya-Qin Chai; Xin-Lu Liu (pp. 855-862).
A novel barium(II) ion-selective polymeric membrane electrode based on a macrocyclic tetrabasic ligand that derived from the reaction of 4,6-diacetylresorcinol and ethylenediamine in the molar ratio 1:1 as neutral carrier was described. The electrode, with optimum membrane composition, exhibited an excellent near-Nernstian response for Ba2+ ion ranging from 3.6 × 10−6 to 1.0 × 10−1 mol/L with a detection limit of 1.9 × 10−6 mol/L and a slope of 29.7 ± 0.2 mV/dec in pH 4.0 nitrate buffer solution at 25 °C. It had a relatively fast response time (10 s), good selectivity towards Ba2+ ion, and could be used over a wide pH range of 2.5–7.5. Then, the response mechanism was discussed in view of UV–visible spectroscopy and the A.C. impedance technique. Finally, the proposed electrode was successfully applied to the detection of Ba2+ ions in real samples as well as in sulfate(II) ions in water.

Keywords: Barium(II) ion-selective electrode; Polymeric membrane; Macrocyclic tetrabasic ligand; Potential response characteristic; Chemical sensors; Ion-selective electrodes; Potentiometry/titrations


A highly selective polymeric membrane barium(II) electrode based on a macrocyclic tetrabasic ligand as neutral carrier by Yu-Hua Ma; Ruo Yuan; Ya-Qin Chai; Xin-Lu Liu (pp. 855-862).
A novel barium(II) ion-selective polymeric membrane electrode based on a macrocyclic tetrabasic ligand that derived from the reaction of 4,6-diacetylresorcinol and ethylenediamine in the molar ratio 1:1 as neutral carrier was described. The electrode, with optimum membrane composition, exhibited an excellent near-Nernstian response for Ba2+ ion ranging from 3.6 × 10−6 to 1.0 × 10−1 mol/L with a detection limit of 1.9 × 10−6 mol/L and a slope of 29.7 ± 0.2 mV/dec in pH 4.0 nitrate buffer solution at 25 °C. It had a relatively fast response time (10 s), good selectivity towards Ba2+ ion, and could be used over a wide pH range of 2.5–7.5. Then, the response mechanism was discussed in view of UV–visible spectroscopy and the A.C. impedance technique. Finally, the proposed electrode was successfully applied to the detection of Ba2+ ions in real samples as well as in sulfate(II) ions in water.

Keywords: Barium(II) ion-selective electrode; Polymeric membrane; Macrocyclic tetrabasic ligand; Potential response characteristic; Chemical sensors; Ion-selective electrodes; Potentiometry/titrations


Protein versus DNA as a marker for peripheral blood mononuclear cell counting by Robert S. Jansen; Hilde Rosing; Jan H. M. Schellens; Jos H. Beijnen (pp. 863-867).
Quantitative analysis of intracellular analytes requires an accurate and precise assay not only for the quantitation of the analytes, but also for the quantitation of the number of cells in which they were determined. In this technical note we compare protein and DNA as markers for the number of peripheral blood mononuclear cells (PBMCs) isolated from whole blood. The protein content of samples was highly influenced by red blood cell contamination and was, therefore, a less suitable marker. The DNA-based method was unaffected by red blood cell contamination and was finally validated over a range from 10 × 106 to 300 × 106 PBMCs/mL. Figure Protein concentration (green) and DNA-based peripheral blood mononuclear cell (PBMC) count (blue) versus cell count obtained using a haemocytometer

Keywords: Bioanalytical methods; Nucleic acids (DNA | RNA); Cell counting; Hoechst 33258


Protein versus DNA as a marker for peripheral blood mononuclear cell counting by Robert S. Jansen; Hilde Rosing; Jan H. M. Schellens; Jos H. Beijnen (pp. 863-867).
Quantitative analysis of intracellular analytes requires an accurate and precise assay not only for the quantitation of the analytes, but also for the quantitation of the number of cells in which they were determined. In this technical note we compare protein and DNA as markers for the number of peripheral blood mononuclear cells (PBMCs) isolated from whole blood. The protein content of samples was highly influenced by red blood cell contamination and was, therefore, a less suitable marker. The DNA-based method was unaffected by red blood cell contamination and was finally validated over a range from 10 × 106 to 300 × 106 PBMCs/mL. Figure Protein concentration (green) and DNA-based peripheral blood mononuclear cell (PBMC) count (blue) versus cell count obtained using a haemocytometer

Keywords: Bioanalytical methods; Nucleic acids (DNA | RNA); Cell counting; Hoechst 33258


Analysis of nonderivatized steroids by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using C70 fullerene as matrix by Gergely Montsko; Alexandra Vaczy; Gabor Maasz; Erzsebet Mernyak; Eva Frank; Csaba Bay; Zalan Kadar; Robert Ohmacht; Janos Wolfling; Laszlo Mark (pp. 869-874).
Neutral steroid hormones are currently analyzed by gas or liquid chromatography/mass spectrometry based methods. Most of the steroid compounds, however, lack volatility and do not contain polar groups, which results in inadequate chromatographic behavior and low ionization efficiency. Derivatization of the steroids to form more volatile, thermostable, and charged products solves this difficulty, but the derivatization of compounds with unknown chemical moieties is not an easy task. In this study, a rapid, high-throughput, sensitive matrix-assisted laser desorption/ionization time-of-flight mass spectrometry method is described using C70 fullerene as a matrix compound. The application of the method is demonstrated for five general sex steroids and for synthetic steroid compounds in both negative and positive ionization modes.

Keywords: Androgens; Endocrinology; Estrogens; Fullerene; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Steroid hormone


Analysis of nonderivatized steroids by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using C70 fullerene as matrix by Gergely Montsko; Alexandra Vaczy; Gabor Maasz; Erzsebet Mernyak; Eva Frank; Csaba Bay; Zalan Kadar; Robert Ohmacht; Janos Wolfling; Laszlo Mark (pp. 869-874).
Neutral steroid hormones are currently analyzed by gas or liquid chromatography/mass spectrometry based methods. Most of the steroid compounds, however, lack volatility and do not contain polar groups, which results in inadequate chromatographic behavior and low ionization efficiency. Derivatization of the steroids to form more volatile, thermostable, and charged products solves this difficulty, but the derivatization of compounds with unknown chemical moieties is not an easy task. In this study, a rapid, high-throughput, sensitive matrix-assisted laser desorption/ionization time-of-flight mass spectrometry method is described using C70 fullerene as a matrix compound. The application of the method is demonstrated for five general sex steroids and for synthetic steroid compounds in both negative and positive ionization modes.

Keywords: Androgens; Endocrinology; Estrogens; Fullerene; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Steroid hormone
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