Analytical and Bioanalytical Chemistry (v.402, #1)

holds the Lucille P. Markey Chair of Biochemistry and Molecular Biology at the Miller School of Medicine of the University of Miami. Previously, she was the Gill Eminent Professor of Chemistry at the University of Kentucky where she had a secondary appointment in the Pharmaceutical Sciences. Dr Daunert’s research interests are in bionanotechnology at the interface between analytical biochemistry, molecular biology, and bioengineering. Her group uses recombinant DNA technology to design new molecular diagnostic tools and biosensors, based on genetically engineered proteins and cells, which have biomedical, environmental, and pharmaceutical applications. The research of the group also focuses on the design of sensing arrays for detection of molecules in small volumes, by use of microfluidics, and in the development of biomaterials for responsive drug-delivery systems.

Meet the contributors by Nicola Oberbeckmann-Winter (15-23).

Changing careers in chemistry by Reiner Salzer (25-28).
retired as Professor of Analytical Chemistry at the Technische Universität Dresden, Germany, in 2007. He obtained his academic degrees from the University of Leipzig, before taking up his appointment at the TU Dresden. His main scientific interests include molecular monitoring for early diagnosis of diseases, integration of biologically active functions into polymers, and electronic media in university education. He is a member of the Norwegian Academy of Science. Professor Salzer is a long-time advisory board member for analytical and spectroscopic journals. He served as President of the Division Analytical Chemistry of the German Chemical Society, and is National Delegate to the Division of Analytical Chemistry of EuCheMS, where he is Head of the Study Group Education. He was elected as Vice-Chairman of the ECTNA Label Committee for the Chemistry Eurobachelor and Chemistry Euromaster and is currently involved in the development of a quality scheme for a Eurodoctorate label.

is the author or coauthor of over 130 research articles, reviews, and book chapters. He is a member of the International Advisory Board of Analytical and Bioanalytical Chemistry. He worked for over 20 years as an analytical chemist for the Chevron Corporation and now runs his own consulting company, Fetzpahs Consulting, in Hercules, CA, USA. His book Career management for chemists—a guide to success in a chemistry career was published by Springer.

is full Research Professor and Deputy Director at IDAEA-CSIC and since 2008 Director of the Catalan Institute for Water Research (ICRA). From 2009, he has been a Visiting Professor at King Saud University, Riyadh, Saudi Arabia. In 2007 he received the Prize King Jaime I from Generalitat of Valencia (Spain) on the Protection of Nature. His research interests include environmental analysis, fate, and behavior of organic pollutants in water, sediment and biota using mass spectrometric techniques, immunoassay, toxic assays and biosensors. In the last few years another interest has been bridging environment and heath risks in the field of emerging contaminants. He is the coordinator of the projects Assessing and Predicting Effects on Water Quantity and Quality in Iberian Rivers (SCARCE) funded by the CONSOLIDER-INGENIO 2010 programme and Orgnaic nanomaterials in the envrionment: characterization, analysis Trojan effects and risks (Nano-Trojan) both funded by the Spanish Ministry of Science and Innovation and of the Project funded by the King Saud University (KSU-VPP-105) Fate and Assessement of Emerging Persitent Organic Pollutants (Perfluorinated compounds and Fullerene nanoparticles): Environmental and Human Health Implications.

Plato’s elements challenge by Juris Meija (35-37).

Solution to the molality–molarity challenge by Mahdi Hashemi; Thomas G. Chasteen (39-39).

Analysis of the bioactivity of magnetically immunoisolated peroxisomes by Yaohua Wang; Thane H. Taylor; Edgar A. Arriaga (41-49).
Peroxisomes produce reactive oxygen species which may participate in biotransformations of innate biomolecules and xenobiotics. Isolating functional peroxisomes with low levels of contaminants would be a useful tool to investigate biotransformations occurring in these organelles that are usually confounded with biotransformations occurring in other co-isolated organelles. Here, we immunoisolate peroxisomes and demonstrate that the impurity level after isolation is low and that peroxisomes retain their biological activity. In this method, an antibody targeting a 70-kDa peroxisomal membrane protein was immobilized to silanized magnetic iron oxide beads (1–4 μm in diameter) coated with Protein A. Peroxisomes from L6 rat myoblast homogenates were magnetically captured, washed, and then analyzed for subcellular composition using enzymatic assays. Based on the ratio of peroxisomal to lysosomal activity, the retained fraction is 70-fold enriched relative to the unretained fraction. Similarly, the ratio of peroxisomal activity to mitochondrial content suggests that the retained fraction is >30-fold enriched relative to the unretained fraction. H2O2 production from the β-oxidation of palmitoyl-CoA demonstrated that the isolated peroxisomal fraction was biologically active. Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) analysis confirmed that the immunopurified fractions were capable of transforming the anticancer drug doxorubicin and the fatty acid analog, BODIPY 500/510 C1C12. Besides its use to investigate peroxisome biotransformations in health and disease, the combination of magnetic immunoisolation with CE-LIF could be widely applicable to investigate subcellular-specific biotransformations of xenobiotics occurring at immunoisolated subcellular compartments. Figure Peroxisomes are immunoisolated using Protein A-coated magnetic beads. The isolated peroxisomes are highly enriched relative to other contaminating organelles. Capillary electrophoretic analysis shows that isolated peroxisomes can biotransform doxorubicin, a common anti-cancer drug.
Keywords: Peroxisome; Fatty acid; Doxorubicin; Magnetic; MEKC; Fluorescence; LIF

Organophosphate and phthalate esters in standard reference material 2585 organic contaminants in house dust by Caroline Bergh; Giovanna Luongo; Stephen Wise; Conny Östman (51-59).
The levels of 22 phthalate diesters (phthalates) and organophosphate triesters (organophosphates) have been investigated in standard reference material 2585 (SRM 2585) “organic contaminants in house dust.” Ultrasonic-assisted solvent extraction and solid-phase extraction on a Florisil adsorbent were used as the extraction and cleanup steps combined with analysis using gas chromatography–tandem mass spectrometry in positive ion chemical ionization mode. Seven phthalates were detected in the concentration range 1–570 μg/g. Di(2-ethylhexyl) phthalate was the major phthalate present at 570 μg/g. Ten organophosphates were detected in SRM 2585. Tris(2-butoxyethyl) phosphate was the predominant organophosphate at 82 μg/g, and nine organophosphates were determined at concentrations ranging from 0.19 to 2.3 μg/g. Five organophosphates were below the method detection limit, of which two were in level with the procedural blank. The applied extraction and cleanup method was evaluated for the analysis of SRM 2585. The extraction yield was ≥99%, except for tris(2-chloroethyl) phosphate (97%) and diethyl phthalate (98.5%). The problem of calibration curvature is addressed, and it is shown that the use of deuterated standards improves the analysis. The concentrations of ten organophosphate esters were determined in SRM 2585, and seven of these were compared with existing data. To our knowledge, this is the first report of the levels of the seven phthalates esters in SRM 2585 “organic contaminants in house dust.”
Keywords: Organophosphate esters; Phthalate esters; Standard reference material 2585; Organic contaminants in house dust

NMR is an invaluable analytical technique that provides structural and chemical information about a molecule without destroying the sample. However, NMR suffers from an inherent lack of sensitivity compared to other popular analytical techniques. This trends article focuses on strategies to increase the sensitivity of NMR using solenoidal microcoil, microstrip, and microslot probes. The role of these reduced-volume receiver coils for detection in hyphenated capillary electrophoresis (CE) and capillary isotachophoresis (cITP) NMR experiments is discussed. Future directions will likely build on work to develop probes containing multiple coils for high-throughput NMR and field-portable instruments.
Keywords: Microcoil NMR; Hyphenated techniques; Microstrip; Microslot

Chemiluminescence (CL) and bioluminescence (BL) are the detection techniques of choice for the development of highly sensitive analytical methods, from immunoassays and nucleic acid hybridization assays to whole-cell biosensors. Nevertheless, basic and applied research on CL and BL aimed at further improving their analytical performance is still very active. This research covers diverse and complementary fields, including (among others) enhancing the light emission efficiency of CL systems, the use of nanomaterials to catalyze or enhance CL/BL reactions, the study of BL proteins to elucidate the color modulation mechanism, the discovery of new BL systems, the production of thermostable BL protein mutants with altered emission spectra, the development of BL imaging techniques to expand our understanding of living systems, and the implementation of CL/BL detection in miniaturized analytical devices. In the near future, we expect even greater diffusion of CL/BL-based analytical methods, especially in portable analytical devices intended for applications ranging from environmental analysis to companion diagnostics for personalized medicine. Figure BL/CL systems and applications (clockwise from top left): in vivo BL imaging, BRET-based system for conformation studies, CL immunohistochemistry in tissue sections, structure of bacterial luciferase, CL systems with different emission wavelenghts, luciferin molecule
Keywords: Bioanalytical methods; Bioluminescence; Biosensors; Chemiluminescence

Hydrodynamic chromatography (HDC) is a liquid chromatographic technique that separates analytes on the basis of their size in solution. Separation can be conducted either in an open tube or in a column packed with inert, nonporous beads. In HDC, larger analytes elute first and smaller ones later, due to preferential sampling of the streamlines of flow in the open tube or in the interstitial medium of the packed column. Because of the low shear rates experienced in HDC, coupled with the wealth of information obtained when employing a multiplicity of detection methods, the technique has experienced a resurgence in recent years in both the particle sizing and macromolecular arenas, where it can provide information on the mutual interdependence of molar mass, size, shape, and compactness. Additionally, microcapillary HDC is also gaining popularity amongst the bioanalytical community, who have employed the technique, inter alia, to separate DNA fragments over a base pair range spanning four orders in magnitude. Here, examples from the literature are used to show how HDC has been applied in each of the aforementioned areas, explaining the information that can be obtained from various detector combinations, and opining on the future of the technique.
Keywords: Hydrodynamic chromatography; Multiple detection; Packed columns; Microcapillaries; Particle sizing; Polymers; DNA

Composite nanoparticles: the best of two worlds by Colleen M. Janczak; Craig A. Aspinwall (83-89).
Nanomaterials have rapidly moved into the mainstream for chemical and biological analysis. Nanoparticle probes enhance signal intensity, increase the chemical and physical stability of the probe, and facilitate surface modification for specific targeting. Unfortunately, common problems are encountered with many nanoparticle probes, e.g., poor solubility, poor biocompatibility, and leakage of encapsulated components, that severely restrict the application of probes to ex vivo samples under carefully controlled conditions. A wide range of recently developed multifunctional nanomaterials are poised to make significant contributions to molecular analysis of biological systems. Composite nanoparticle geometries, including composites, hybrids, and core–shell nanoparticles prepared using two or more materials, e.g., silica/inorganic, silica/polymer, or polymer/inorganic combinations, offer improved solubility, easier functionalization, and decreased toxicity compared with the related single-component materials. Furthermore, composite nanomaterials present substantial signal amplification, and improved multiplexing for higher-sensitivity and higher-resolution measurements. Further development and integration of composite nanomaterials into the quantitative sciences will play a key role in the future of functional probes for imaging, quantitative analysis, and biological manipulation. Figure 1 Multifunctional nanoparticle geometries, including core-shell and composite architectures, are increasing used for biological and chemical analysis.
Keywords: Nanoparticles; Silica; Polymer; Nanomaterials; Hybrid; Composite

Towards compound-independent calibration for organic compounds using online isotope dilution mass spectrometry by Sergio Cueto Díaz; Jorge Ruiz Encinar; Alfredo Sanz-Medel; José Ignacio García Alonso (91-97).
Isotope dilution mass spectrometry (IDMS) can be considered a primary measurement method directly traceable to the International System of Units (SI). This measurement technique is increasingly employed in routine laboratories, owing to its unequalled analytical performance, precision and ease of accreditation. Unfortunately, for the adequate application of IDMS, several isotopically labelled standards, corresponding to the compounds of interest, are required. Additionally, when the enriched isotope is continuously added after a chromatographic separation, and an elemental ion source is used, it allows quantification of the different analytes being eluted from the column without requiring specific standards for each compound (online IDMS). In this article, we discuss how the traditional applicability of online IDMS for elemental speciation can be dramatically expanded by using carbon isotope tracers, oxidation or combustion reactions and a conventional molecular ion source. With such a strategy every carbon-containing compound being eluted from a chromatography system can be quantified without the need for specific standards as long as quantitative combustion/oxidation and complete elution occur. So far, only gas chromatography–combustion–mass spectrometry applications have been described, but recent results indicate the great possibilities of extending this novel approach to the quantification of organic compounds after separation by liquid chromatography.
Keywords: Isotope dilution mass spectrometry; Compound-independent calibration; Organic compounds; Chromatography

Microchip-based cellular biochemical systems for practical applications and fundamental research: from microfluidics to nanofluidics by Yan Xu; Kihoon Jang; Tadahiro Yamashita; Yo Tanaka; Kazuma Mawatari; Takehiko Kitamori (99-107).
By combining cell technology and microchip technology, innovative cellular biochemical tools can be created from the microscale to the nanoscale for both practical applications and fundamental research. On the microscale level, novel practical applications taking advantage of the unique capabilities of microfluidics have been accelerated in clinical diagnosis, food safety, environmental monitoring, and drug discovery. On the other hand, one important trend of this field is further downscaling of feature size to the 101–103 nm scale, which we call extended-nano space. Extended-nano space technology is leading to the creation of innovative nanofluidic cellular and biochemical tools for analysis of single cells at the single-molecule level. As a pioneering group in this field, we focus not only on the development of practical applications of cellular microchip devices but also on fundamental research to initiate new possibilities in the field. In this paper, we review our recent progress on tissue reconstruction, routine cell-based assays on microchip systems, and preliminary fundamental method for single-cell analysis at the single-molecule level with integration of the burgeoning technologies of extended-nano space. Figure By combination of cell technology and microchip technology, innovative cellular biochemical tools can be created from the microscale to the nanoscale, for both practical applications and fundamental research. The image demonstrates a concept of analysis of a single cell at the singlemolecule level on a microchip with integration of extended-nano (101–103 nm) space.
Keywords: Extended-nano space; Tissue engineering; Cell-based assay; DNA detection; Single cell; Single molecule

Optical fibre gratings as tools for chemical and biochemical sensing by F. Baldini; M. Brenci; F. Chiavaioli; A. Giannetti; C. Trono (109-116).
Optical fibre gratings have recently been suggested as optical platforms for chemical and biochemical sensing. On the basis of the measurement of refractive index changes induced by a chemical and biochemical interaction in the transmission spectrum along the fibres, they are proposed as a possible alternative to the other label-free optical approaches, such as surface plasmon resonance and optical resonators. The combination of the use of optical fibres with the fact that the signal modulation is spectrally encoded offers multiplexing and remote measurement capabilities which the other technology platforms are not able to or can hardly offer. The fundamentals of the different types of optical fibre gratings are described and the performances of the chemical and biochemical sensors based on this approach are reviewed. Advantages and limitations of optical fibre gratings are considered, with a look at new perspectives for their utilization in the field. Figure Basic configurations of optical fibre gratings: (a) standard fibre Bragg grating; (b) standard long period grating; (c) tilted fibre Bragg grating; (d) etched fibre Bragg grating.
Keywords: Optical grating; Long-period grating; Fibre Bragg grating; Chemical sensing; Biochemical sensing

Biosensors for the detection of waterborne pathogens by John T. Connelly; Antje J. Baeumner (117-127).
Waterborne bacterial, viral and parasitic pathogens are a global health concern and their rapid and specific detection in contaminated potable water is of utmost importance. Biosensors using a variety of biorecognition molecules and transduction methodologies have been reported, and have the potential to enable highly sensitive detection of the analyte of interest in a short time with high specificity. However, there are several obstacles to the detection of waterborne pathogens—they tend to be present at very low concentrations in the environment and environmental samples contain numerous inhibitors of enzymatic reactions and interfering organisms and particulates. Here we present a review of the current state of biosensor technology with regard to the improvements needed over standard detection methods and the challenges presented by real environmental samples. Further, we identify future areas of focus necessary to realize novel detection devices capable of supplanting the gold standards of today.
Keywords: Biosensor; Waterborne; Pathogen; Microfluidic

DNA-templated fluorescent silver nanoclusters by Bingyan Han; Erkang Wang (129-138).
In this review, we discuss the synthesis and applications of DNA-templated fluorescent silver nanoclusters in aqueous solution. Various oligonucleotide sequences or conformations have been utilized to synthesize silver nanoclusters with excellent fluorescence properties. The range of applications has expanded greatly, from live cell staining and the detection of metal ions and small biomolecules to the detection of DNA or proteins.
Keywords: Silver nanoclusters; Oligonucleotides; DNA; Fluorescence

Challenges and trends in the determination of selected chemical contaminants and allergens in food by Rudolf Krska; Adam Becalski; Eric Braekevelt; Terry Koerner; Xu-Liang Cao; Robert Dabeka; Samuel Godefroy; Ben Lau; John Moisey; Dorothea F. K. Rawn; Peter M. Scott; Zhongwen Wang; Don Forsyth (139-162).
This article covers challenges and trends in the determination of some major food chemical contaminants and allergens, which—among others—are being monitored by Health Canada’s Food Directorate and for which background levels in food and human exposure are being analyzed and calculated. Eleven different contaminants/contaminant groups and allergens have been selected for detailed discussion in this paper. They occur in foods as a result of: use as a food additive or ingredient; processing-induced reactions; food packaging migration; deliberate adulteration; and/or presence as a chemical contaminant or natural toxin in the environment. Examples include acrylamide as a food-processing-induced contaminant, bisphenol A as a food packaging-derived chemical, melamine and related compounds as food adulterants and persistent organic pollutants, and perchlorate as an environmental contaminant. Ochratoxin A, fumonisins, and paralytic shellfish poisoning toxins are examples of naturally occurring toxins whereas sulfites, peanuts, and milk exemplify common allergenic food additives/ingredients. To deal with the increasing number of sample matrices and analytes of interest, two analytical approaches have become increasingly prevalent. The first has been the development of rapid screening methods for a variety of analytes based on immunochemical techniques, utilizing ELISA or surface plasmon resonance technology. The second is the development of highly sophisticated multi-analyte methods based on liquid chromatography coupled with multiple-stage mass spectrometry for identification and simultaneous quantification of a wide range of contaminants, often with much less requirement for tedious cleanup procedures. Whereas rapid screening methods enable testing of large numbers of samples, the multi analyte mass spectrometric methods enable full quantification with confirmation of the analytes of interest. Both approaches are useful when gathering surveillance data to determine occurrence and background levels of both recognized and newly identified contaminants in foods in order to estimate human daily intake for health risk assessment.
Keywords: Food safety; Chemical contaminants; Acrylamide; Bisphenol A; Melamine; Perchlorate; Sulfites; Persistent organic pollutants; Mycotoxins; Phycotoxins; Allergens

Spectroscopic methods in gas hydrate research by Florian Rauh; Boris Mizaikoff (163-173).
Gas hydrates are crystalline structures comprising a guest molecule surrounded by a water cage, and are particularly relevant due to their natural occurrence in the deep sea and in permafrost areas. Low molecular weight molecules such as methane and carbon dioxide can be sequestered into that cage at suitable temperatures and pressures, facilitating the transition to the solid phase. While the composition and structure of gas hydrates appear to be well understood, their formation and dissociation mechanisms, along with the dynamics and kinetics associated with those processes, remain ambiguous. In order to take advantage of gas hydrates as an energy resource (e.g., methane hydrate), as a sequestration matrix in (for example) CO2 storage, or for chemical energy conservation/storage, a more detailed molecular level understanding of their formation and dissociation processes, as well as the chemical, physical, and biological parameters that affect these processes, is required. Spectroscopic techniques appear to be most suitable for analyzing the structures of gas hydrates (sometimes in situ), thus providing access to such information across the electromagnetic spectrum. A variety of spectroscopic methods are currently used in gas hydrate research to determine the composition, structure, cage occupancy, guest molecule position, and binding/formation/dissociation mechanisms of the hydrate. To date, the most commonly applied techniques are Raman spectroscopy and solid-state nuclear magnetic resonance (NMR) spectroscopy. Diffraction methods such as neutron and X-ray diffraction are used to determine gas hydrate structures, and to study lattice expansions. Furthermore, UV-vis spectroscopic techniques and scanning electron microscopy (SEM) have assisted in structural studies of gas hydrates. Most recently, waveguide-coupled mid-infrared spectroscopy in the 3–20 μm spectral range has demonstrated its value for in situ studies on the formation and dissociation of gas hydrates. This comprehensive review summarizes the importance of spectroscopic analytical techniques to our understanding of the structure and dynamics of gas hydrate systems, and highlights selected examples that illustrate the utility of these individual methods. Figure Spectroscopic techniques play an increasingly important role in gas hydrate research.
Keywords: Geochemistry; Geology; Oceanography; Spectroscopy; Instrumentation

Trace analysis of environmental matrices by large-volume injection and liquid chromatography–mass spectrometry by Francesco Busetti; Will J. Backe; Nina Bendixen; Urs Maier; Benjamin Place; Walter Giger; Jennifer A. Field (175-186).
The time-honored convention of concentrating aqueous samples by solid-phase extraction (SPE) is being challenged by the increasingly widespread use of large-volume injection (LVI) liquid chromatography–mass spectrometry (LC–MS) for the determination of traces of polar organic contaminants in environmental samples. Although different LVI approaches have been proposed over the last 40 years, the simplest and most popular way of performing LVI is known as single-column LVI (SC-LVI), in which a large-volume of an aqueous sample is directly injected into an analytical column. For the purposes of this critical review, LVI is defined as an injected sample volume that is ≥10% of the void volume of the analytical column. Compared with other techniques, SC-LVI is easier to set up, because it requires only small hardware modifications to existing autosamplers and, thus, it will be the main focus of this review. Although not new, SC-LVI is gaining acceptance and the approach is emerging as a technique that will render SPE nearly obsolete for many environmental applications. In this review, we discuss: the history and development of various forms of LVI; the critical factors that must be considered when creating and optimizing SC-LVI methods; and typical applications that demonstrate the range of environmental matrices to which LVI is applicable, for example drinking water, groundwater, and surface water including seawater and wastewater. Furthermore, we indicate direction and areas that must be addressed to fully delineate the limits of SC-LVI.
Keywords: Large-volume injection; Direct injection; Liquid chromatography; LC–MS–MS; Soil; Solid-phase extraction; Sample preparation; Water; Wastewater

Aptamer-incorporated hydrogels for visual detection, controlled drug release, and targeted cancer therapy by Jun Liu; Huixia Liu; Huaizhi Kang; Michael Donovan; Zhi Zhu; Weihong Tan (187-194).
Hydrogels are water-retainable materials, made from cross-linked polymers, that can be tailored to applications in bioanalysis and biomedicine. As technology advances, an increasing number of molecules have been used as the components of hydrogel systems. However, the shortcomings of these systems have prompted researchers to find new materials that can be incorporated into them. Among all of these emerging materials, aptamers have recently attracted substantial attention because of their unique properties, for example biocompatibility, selective binding, and molecular recognition, all of which make them promising candidates for target-responsive hydrogel engineering. In this work, we will review how aptamers have been incorporated into hydrogel systems to enable colorimetric detection, controlled drug release, and targeted cancer therapy.
Keywords: Biosensors; Nanoparticles/nanotechnology; Polymers; Hydrogels; Aptamers; Drug delivery

This paper reviews scientific contributions on the identification and/or quantification of metabolites of drugs of abuse in in vitro assays or various body samples using hyphenated mass spectrometry. Gas chromatography–mass spectrometry (GC-MS) as well as liquid chromatography–mass spectrometry (LC-MS) approaches are considered and discussed if they have been reported in the last five years and are relevant to clinical and forensic toxicology or doping control. Workup and artifact formation are discussed, and typical examples of studies of the metabolism of designer drugs, doping agents, herbal drugs, and synthetic cannabinoids are provided. Procedures for quantifying metabolites in body samples for pharmacokinetic studies or in enzyme incubations for enzyme kinetic studies are also reviewed. In conclusion, the reviewed papers showed that both GC-MS and LC-MS still have important roles to play in research into the metabolism of drugs of abuse, including doping agents.
Keywords: Drugs of abuse; Doping; Mass spectrometry; Liquid chromatography; Gas chromatography; Metabolism

Natural and artificial ion channels for biosensing platforms by L. Steller; M. Kreir; R. Salzer (209-230).
The single-molecule selectivity and specificity of the binding process together with the expected intrinsic gain factor obtained when utilizing flow through a channel have attracted the attention of analytical chemists for two decades. Sensitive and selective ion channel biosensors for high-throughput screening are having an increasing impact on modern medical care, drug screening, environmental monitoring, food safety, and biowarefare control. Even virus antigens can be detected by ion channel biosensors. The study of ion channels and other transmembrane proteins is expected to lead to the development of new medications and therapies for a wide range of illnesses. From the first attempts to use membrane proteins as the receptive part of a sensor, ion channels have been engineered as chemical sensors. Several other types of peptidic or nonpeptidic channels have been investigated. Various gating mechanisms have been implemented in their pores. Three technical problems had to be solved to achieve practical biosensors based on ion channels: the fabrication of stable lipid bilayer membranes, the incorporation of a receptor into such a structure, and the marriage of the modified membrane to a transducer. The current status of these three areas of research, together with typical applications of ion-channel biosensors, are discussed in this review.
Keywords: Biosensor; Ion channel; Lipid membrane; Planar patch clamp; High throughput; Drug sensing

Hydrophilic interaction liquid chromatography (HILIC) provides an alternative approach to effectively separate small polar compounds on polar stationary phases. The purpose of this work was to review the options for the characterization of HILIC stationary phases and their applications for separations of polar compounds in complex matrices. The characteristics of the hydrophilic stationary phase may affect and in some cases limit the choices of mobile phase composition, ion strength or buffer pH value available, since mechanisms other than hydrophilic partitioning could potentially occur. Enhancing our understanding of retention behavior in HILIC increases the scope of possible applications of liquid chromatography. One interesting option may also be to use HILIC in orthogonal and/or two-dimensional separations. Bioapplications of HILIC systems are also presented. Figure
Keywords: Hydrophilic interaction liquid chromatography; Stationary phase; Separation mechanism; Bioapplication

Phosphorylation site localization in peptides by MALDI MS/MS and the Mascot Delta Score by Simone Lemeer; Elena Kunold; Susan Klaeger; Monika Raabe; Mark W. Towers; Emmanuelle Claudes; Tabiwang N. Arrey; Kerstin Strupat; Henning Urlaub; Bernhard Kuster (249-260).
Owing to its broad biological significance, the large-scale analysis of protein phosphorylation is more and more getting into the focus of proteomic research. Thousands of phosphopeptides can nowadays be identified using state-of-the-art tandem mass spectrometers in conjunction with sequence database searching, but localizing the phosphate group to a particular amino acid in the peptide sequence is often still difficult. Using 180 individually synthesized phosphopeptides with precisely known phosphorylation sites (p-sites), we have assessed the merits of the Mascot Delta Score (MD score) for the assignment of phosphorylation sites from tandem mass spectra (MS/MS) generated on four different matrix-assisted laser desorption ionization (MALDI) mass spectrometers including tandem time-of-flight (TOF/TOF), quadrupole time-of-flight, and ion trap mass analyzers. The results show that phosphorylation site identification is generally possible with false localization rates of about 10%. However, a comparison to previous work also revealed that phosphorylation site determination by MALDI MS/MS is less accurate than by ESI-MS/MS particularly if several and/or adjacent possible phosphorylation acceptor sites exist in a peptide sequence. We are making the tandem MS spectra and phosphopeptide collection available to the community so that scientists may adapt the MD scores reported here to their analytical environment and so that informatics developers may integrate the MD score into proteomic data analysis pipelines.
Keywords: Mass spectrometry; Phosphorylation; Proteomics; False localization rate

Liquid sampling–atmospheric pressure glow discharge (LS-APGD) ionization source for elemental mass spectrometry: preliminary parametric evaluation and figures of merit by C. Derrick Quarles Jr.; Anthony J. Carado; Charles J. Barinaga; David W. Koppenaal; R. Kenneth Marcus (261-268).
A new, low-power ionization source for the elemental analysis of aqueous solutions has recently been described. The liquid sampling–atmospheric pressure glow discharge (LS-APGD) source operates at relatively low currents (<20 mA) and solution flow rates (<50 μL min−1), yielding a relatively simple alternative for atomic mass spectrometry applications. The LS-APGD has been interfaced to what is otherwise an organic, LC-MS mass analyzer, the Thermo Scientific Exactive Orbitrap without any modifications, other than removing the electrospray ionization source supplied with that instrument. A glow discharge is initiated between the surface of the test solution exiting a glass capillary and a metallic counter electrode mounted at a 90° angle and separated by a distance of ~5 mm. As with any plasma-based ionization source, there are key discharge operation and ion sampling parameters that affect the intensity and composition of the derived mass spectra, including signal-to-background ratios. We describe here a preliminary parametric evaluation of the roles of discharge current, solution flow rate, argon sheath gas flow rate, and ion sampling distance as they apply on this mass analyzer system. A cursive evaluation of potential matrix effects due to the presence of easily ionized elements indicate that sodium concentrations of up to 50 μg mL−1 generally cause suppressions of less than 50%, dependant upon the analyte species. Based on the results of this series of studies, preliminary limits of detection (LOD) have been established through the generation of calibration functions. While solution-based concentration LOD levels of 0.02–2 μg mL−1 are not impressive on the surface, the fact that they are determined via discrete 5 μL injections leads to mass-based detection limits at picogram to single-nanogram levels. The overhead costs associated with source operation (10 W d.c. power, solution flow rates of <50 μL min−1, and gas flow rates <10 mL min−1) are very attractive. While further optimization in the source design is suggested here, it is believed that the LS-APGD ion source may present a practical alternative to inductively coupled plasma sources typically employed in elemental mass spectrometry.
Keywords: Liquid sampling–atmospheric pressure glow discharge; Ionization source; Microplasma; Mass spectrometry

Direct μ-flow injection isotope dilution ICP-MS for the determination of heavy metals in oil samples by Jörg Bettmer; Jens Heilmann; Daniel J. Kutscher; Alfredo Sanz-Medel; Klaus G. Heumann (269-275).
The determination of trace elements in oil samples and their products is of high interest as their presence significantly affects refinery processes and the environment by possible impact of their combustion products. In this context, inductively coupled plasma mass spectrometry (ICP-MS) plays an important role due to its outstanding analytical properties in the quantification of trace elements. In this work, we present the accurate and precise determination of selected heavy metals in oil samples by making use of the combination of μ-flow direct injection and isotope dilution ICP-MS (ICP-IDMS). Spike solutions of 62Ni, 97Mo, 117Sn and 206Pb were prepared in an organic solvent, mixed directly with the diluted oil samples and tested to be fit for purpose for the intended ID approach. The analysis of real samples revealed strong matrix effects affecting the ICP-MS sensitivity, but not the isotope ratio measurements, so that accurate results are obtained by ICP-IDMS. Typical relative standard deviations were about 15% for peak area and peak height measurements, whereas the isotope ratios were not significantly affected (RSD < 2%). The developed method was validated by the analysis of a metallo-organic multi-element standard (SCP-21, typically applied as a calibration standard) and the standard reference material SRM1084a (wear metals in lubricating oil). The obtained results were in excellent agreement with the certified values (recoveries between 98% and 102%), so the proposed methodology of combining μ-flow direct injection and ICP-IDMS can be regarded as a new tool for the matrix-independent, multi-element and reliable determination of trace elements in oil and related organic liquids. Figure Determination of heavy metals in oil samples by ICP-IDMS
Keywords: Inductively coupled plasma–mass spectrometry (ICP-MS); Trace elements; Isotope dilution analysis; μ-Flow injection; Oil samples; Direct analysis

The use of V(IV) complexes as insulin-enhancing agents has been increasing during the last decade. Among them, 3-hydroxy-2-methyl-4-pyrone and 2-ethyl-3-hydroxy-4-pyrone (maltol and ethyl maltol, respectively) have proven to be especially suitable as ligands for vanadyl ions. In fact, they have passed phase I and phase II clinical trials, respectively. However, the mechanism through which those drugs exert their insulin-mimetic properties is still not fully understood. Thus, the aim of this study is to obtain an integrated picture of the absorption, biodistribution and insulin-mimetic properties of the bis(maltolato)oxovanadium (IV) (BMOV) in streptozotocin-induced hyperglycaemic rats. For this purpose, BMOV hypoglycaemic properties were evaluated by monitoring both the circulating glucose and the glycohemoglobin, biomarkers of diabetes mellitus. In both cases, the results were drug concentration dependent. Using doses of vanadium at 3 mg/day, it was possible to reduce the glycaemia of the diabetic rats to almost control levels. BMOV absorption experiments have been conducted by intestinal perfusion revealing that approximately 35% of V is absorbed by the intestinal cells. Additionally, the transport of the absorbed vanadium (IV) by serum proteins was studied. For this purpose, a speciation strategy using high-performance liquid chromatography (HPLC) for separation and inductively coupled serum mass spectrometry, ICP-MS, for detection has been employed. The obtained HPLC-ICP-MS results, confirmed by MALDI-MS data, showed evidence that V, administered orally, is uniquely bound to transferrin in rat serum.
Keywords: Vanadium; Transferrin; Insulin mimetic; HPLC; Mass spectrometry; ICP-MS; MALDI-TOF

Microanalytical isotope ratio measurements and elemental mapping using laser ablation ICP-MS for tissue thin sections: zinc tracer studies in rats by Dagmar S. Urgast; Ou Ou; Margaret-Jane Gordon; Andrea Raab; Graeme F. Nixon; In-Sook Kwun; John H. Beattie; Jörg Feldmann (287-297).
The kinetics of zinc absorption, metabolism and excretion is extensively studied by nutritionists. Stable isotopes of zinc can be used to identify body zinc compartments that have different turnover kinetics. Since the compartments might belong to physiological subsections of different organs, there is a need for microsampling analysis to determine isotope ratios of the trace element zinc in tissue samples. Here, we study the feasibility to use laser ablation coupled to quadrupole ICP-MS for the determination of zinc tracers given to rats at different time points with the aim to generate isotope ratio bioimages of heart tissue. A double tracer (70Zn and 67Zn) experiment with rats was designed to label the exchangeable zinc pool as well as the stable zinc pool. The isotope ratios determined by laser ablation ICP-MS were evaluated by additional measurements of tissue digests. Accumulated tracers which made up more than 0.1% of total zinc could be identified in the tissues of the treated rats. It was established that at least 50 measurements from the microsampling were necessary to distinguish between controls and a tracer treated rat resulting in reduced resolution of the bioimage. With the parameters used, features in the tissue thin sections of at least 250 μm2 in size are necessary to detect the incorporation of a tracer. When different time points have to be measured, higher precisions are required and therefore a larger area needs to be ablated (1 mm2). Using the bioimages and pool measurements from one physiological feature, it was possible to show that the aorta cell walls incorporate the zinc tracer at the different time points.
Keywords: LA-ICP-MS; Zinc; Bioimaging; Tracer; Rat; Accumulation; Isotope ratio; Mapping; Thin sections; Heart; Liver; Muscle

New insights on proteomics of transgenic soybean seeds: evaluation of differential expressions of enzymes and proteins by Herbert S. Barbosa; Sandra C. C. Arruda; Ricardo A. Azevedo; Marco A. Z. Arruda (299-314).
This work reports the evaluation of differentially expressed enzymes and proteins from transgenic and nontransgenic soybean seeds. Analysis of malondialdehyde, ascorbate peroxidase (EC, glutathione reductase (EC, and catalase (EC revealed higher levels (29.8, 30.6, 71.4, and 35.3%, respectively) in transgenic seeds than in nontransgenic seeds. Separation of soybean seed proteins was done by two-dimensional polyacrylamide gel electrophoresis, and 192 proteins were identified by matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) and electrospray ionization (ESI) QTOF MS. Additionally, the enzyme CP4 EPSPS, involved in the genetic modification, was identified by enzymatic digestions using either trypsin or chymotrypsin and ESI-QTOF MS/MS for identification. From the proteins identified, actin fragment, cytosolic glutamine synthetase, glycinin subunit G1, and glycine-rich RNA-binding protein were shown to be differentially expressed after analysis using the two-dimensional difference gel electrophoresis technique, and applying a regulator factor of 1.5 or greater. Figure Application of in gel electrophoretic techniques for evaluation of transgenic soybean seeds from differential expressions of enzymes and proteins
Keywords: Two-dimensional difference gel electrophoresis; Mass spectrometry; Reactive oxygen species; Genetically modified organisms

Micro-total analysis system for virus detection: microfluidic pre-concentration coupled to liposome-based detection by John T. Connelly; Sowmya Kondapalli; Marc Skoupi; John S. L. Parker; Brian J. Kirby; Antje J. Baeumner (315-323).
An integrated microfluidic biosensor is presented that combines sample pre-concentration and liposome-based signal amplification for the detection of enteric viruses present in environmental water samples. This microfluidic approach overcomes the challenges of long assay times of cell culture-based methods and the need to extensively process water samples to eliminate inhibitors for PCR-based methods. Here, viruses are detected using an immunoassay sandwich approach with the reporting antibodies tagged to liposomes. Described is the development of the integrated device for the detection of environmentally relevant viruses using feline calicivirus (FCV) as a model organism for human norovirus. In situ fabricated nanoporous membranes in glass microchannels were used in conjunction with electric fields to achieve pre-concentration of virus–liposome complexes and therefore enhance the antibody–virus binding efficiency. The concentrated complexes were eluted to a detection region downstream where captured liposomes were lysed to release fluorescent dye molecules that were then quantified using image processing. This system was compared to an optimized electrochemical liposome-based microfluidic biosensor without pre-concentration. The limit of detection of FCV of the integrated device was at 1.6 × 105 PFU/mL, an order of magnitude lower than that obtained using the microfluidic biosensor without pre-concentration. This significant improvement is a key step toward the goal of using this integrated device as an early screening system for viruses in environmental water samples.
Keywords: Biosensor; Microfluidic; Pre-concentration; FCV; Liposomes

Hydrodynamic focusing—a versatile tool by Joel P. Golden; Gusphyl A. Justin; Mansoor Nasir; Frances S. Ligler (325-335).
The control of hydrodynamic focusing in a microchannel has inspired new approaches for microfluidic mixing, separations, sensors, cell analysis, and microfabrication. Achieving a flat interface between the focusing and focused fluids is dependent on Reynolds number and device geometry, and many hydrodynamic focusing systems can benefit from this understanding. For applications where a specific cross-sectional shape is desired for the focused flow, advection generated by grooved structures in the channel walls can be used to define the shape of the focused flow. Relative flow rates of the focused flow and focusing streams can be manipulated to control the cross-sectional area of the focused flows. This paper discusses the principles for defining the shape of the interface between the focused and focusing fluids and provides examples from our lab that use hydrodynamic focusing for impedance-based sensors, flow cytometry, and microfabrication to illustrate the breadth of opportunities for introducing new capabilities into microfluidic systems. We evaluate each example for the advantages and limitations integral to utilization of hydrodynamic focusing for that particular application.
Keywords: Hydrodynamic focusing; Sheath flow; Microfluidics; Conductivity; Flow focusing; Reynolds number

The ligand epitope map and the effect of nonspecific binding is assessed for lidocaine binding to α1-acid glycoprotein using the saturation transfer difference nuclear magnetic resonance experiment performed as a function of the ligand/protein ratio. The experimental design tested two different approaches for preparing solutions with various ligand/protein ratios; holding the protein concentration constant and increasing the ligand concentration; and holding the ligand concentration constant while decreasing the protein concentration. Nonspecific binding effects were more prevalent in experiments in which the ligand concentration was increased, although spectra with higher signal-to-noise ratios were obtained under these conditions. The epitope map determined for achiral lidocaine is compared with previously determined results for the (R)- and (S)-enantiomers of propranolol. The weaker binding affinity of lidocaine may be partially attributed to steric hindrance by the lidocaine N-ethyl groups which may prevent close contact of the lidocaine amine with the negatively charged amino acids at the apex of the protein binding pocket.
Keywords: Nonspecific binding; Lidocaine; α1-Acid glycoprotein; AGP; Epitope mapping; STD; NMR

Label-free quantification of cystatin C as an improved marker for renal failure by Oliver Bleher; Markus Ehni; Günter Gauglitz (349-356).
A label-free biosensor has been developed, allowing quantification of cystatin C in human serum. This was achieved by using reflectometric interference spectroscopy as detection method. Cystatin C is a small serum protein that allows detection of renal failure more reliably than established parameters as creatinine. The protein was immobilized on the surface of a glass transducer, forming the sensitive layer of the sensor chip. Based on a binding-inhibition assay, two different types of monoclonal cystatin C antibodies were compared, by their behavior and their obtained working range in buffer and serum as matrix. Both antibodies allowed quantification of the protein in serum as matrix within the required clinical ranges of 0.53–1.02 mg/L. Detected recovery rates are in a range between 84.8% and 116.1%. The developed sensor shows high inner chip reproducibility and low cross-sensitivity. Figure Binding curves with 4 mg/L of antibody (A), inhibited with 0.4/0.8/4 mg/L cystatin C. A constant concentration of 4 mg/L antibody (A) is incubated with 0.4/0.8/4 mg/L cystatin C. Unspecific binding is blocked with an ovalbumin (OVA) solution of 1 mg/mL in PBS. The surface is regenerated by a SDS solution of 0.5%/pH 2
Keywords: Reflectometric interference spectroscopy (RIfS); Optical sensors; Label-free detection; Renal failure; Cystatin C; Biosensor

Absolute quantification of free glutathione and cysteine in aquatic insects using isotope dilution and selected reaction monitoring by Christopher M. Shuford; Monica D. Poteat; David B. Buchwalter; David C. Muddiman (357-366).
A simple and robust isotope dilution mass spectrometry-based assay was developed for the determination of free cysteine and glutathione (GSH) in aquatic insects. Several experimental parameters were evaluated and optimized to provide specific and sensitive detection of both compounds by in situ derivatization with N-ethylmaleimide followed by acid alkylation quenching and reverse-phased liquid chromatography coupled with selected reaction monitoring. For both targets, the assay was evaluated over a concentration range of 0.313 to 320 μM and was demonstrated to have a quantitative dynamic range spanning nearly three orders of magnitude, with lower limits of quantification being 0.330 μM for GSH and 0.370 μM for cysteine. Additionally, measurements were observed to be highly reproducible over the course of several days. When applied to the analysis of four different species of insects, large biological variation between and within species was observed. Different feeding regimens were also tested within two species of insects but statistical comparisons revealed no significant difference in the levels of either compound. Figure Free forms of glutathione and cysteine are stabilized by chemical modification in the homogenates of various aquatic insects. Use stable isotope-labeled internal standards, the modified antioxidants are quantified using RPLC and selected reaction monitoring
Keywords: Glutathione; Cysteine; Selected reaction monitoring; Absolute quantification; Isotope dilution

The determination of total deoxyribonucleic acid (DNA) concentration is of great importance in many biological and bio-medical analyses. The quantification of DNA is traditionally performed by UV spectroscopy; however the results can be affected greatly by the sample matrix. The proposed method quantifies phosphorus in digested calf thymus DNA and human DNA by high performance liquid chromatography (HPLC) combined with inductively coupled plasma mass spectrometry (ICP-MS). The method presented showed excellent baseline separation between all four DNA mono-nucleotides and 5′UMP. The ability of LC-ICP-MS to provide an internal check that only DNA derived phosphorus was counted in the assay was demonstrated by establishing a mass balance between the total phosphorous signal from undigested DNA and that from the speciated DNA. Column recoveries ranging from 95% to 99% for phosphorus resulted in a mass balance of 95% ± 0.5% for standard nucleotides, determined by LC-ICP-MS, compared to total DNA determined by flow injection coupled to ICP-MS (FI-ICP-MS). The method for quantification was validated by analysis of NIST SRM 2,372; a total speciated DNA recovery of 52.1 ng/μL, compared with an expected value of 53.6 ng/μL, was determined by external calibration. From repeat measurements, a mass balance of 97% ± 0.5% for NIST DNA was achieved. The method limits of detection for individual nucleotides were determined between 0.8 and 1.7 μg L−1 (31P) for individual nucleotides by LC-ICP-MS, and 360 ng L−1 for 5′AMP by direct nebulisation.
Keywords: Quantification; DNA; ICP-MS; Mass balance; Nucleotides

Time-resolved chloroquine-induced relaxation of supercoiled plasmid DNA by Marek Mahut; Michael Leitner; Andreas Ebner; Michael Lämmerhofer; Peter Hinterdorfer; Wolfgang Lindner (373-380).
Herein, we report on the in vitro change of DNA conformation of plasmids bound to a 3-aminopropyl-modified mica surface and monitoring the events by atomic force microscopy (AFM) imaging under near physiological conditions. In our study, we used an intercalating drug, chloroquine, which is known to decrease the twist of the double helix and thus altered the conformation of the whole DNA. During our experiments, a chloroquine solution was added while imaging a few highly condensed plasmid nanoparticles in solution. AFM images recorded after the drug addition clearly show a time-resolved relaxation of these bionanoparticles into a mixture of loose DNA strands. Figure Condensed plasmid DNA nanoparticles are relaxed in aqueous environment by addition of chloroquine. This process, monitored by atomic force microscopy in liquid phase, reveals different structural behavior depending on the surface modification.
Keywords: Atomic force microscopy; DNA condensation; Plasmid DNA; Chloroquine; Bionanoparticles; Intercalation

Surface plasmon resonance biosensor for the detection of VEGFR-1—a protein marker of myelodysplastic syndromes by Kristýna Pimková; Markéta Bocková; Kateřina Hegnerová; Jiří Suttnar; Jaroslav Čermák; Jiří Homola; Jan E. Dyr (381-387).
The surface plasmon resonance (SPR) biosensor system with dispersionless microfluidics for the direct and label-free detection of a soluble vascular endothelial growth factor receptor (sVEGFR-1) is described. The detection approach takes advantage of an affinity interaction between sVEGFR-1 and its ligand, vascular endothelial growth factor (VEGF-A), which is covalently immobilized on the surface of the SPR sensor. The ability of the immobilized VEGF-A to specifically bind the sVEGFR-1 receptor is demonstrated in a buffer. The detection of sVEGFR-1 in 2% human blood plasma is carried out by using the sequential injection approach. The detection limit of 25 ng/mL is achieved. In addition, we demonstrate that the functional surface of the sensor can be regenerated for repeated use. Figure The detection of MDS protein marker, sVEGFR-1 (a soluble form of angiogenic receptor), via its physiological counterpart VEGF-A immobilized on the surface of an SPR sensor.
Keywords: Surface plasmon resonance; Myelodysplastic syndromes; Vascular endothelial growth factor; Protein markers

A chemiluminescence sensor array for discriminating natural sugars and artificial sweeteners by Weifen Niu; Hao Kong; He Wang; Yantu Zhang; Sichun Zhang; Xinrong Zhang (389-395).
In this paper, we report a chemiluminescence (CL) sensor array based on catalytic nanomaterials for the discrimination of ten sweeteners, including five natural sugars and five artificial sweeteners. The CL response patterns (“fingerprints”) can be obtained for a given compound on the nanomaterial array and then identified through linear discriminant analysis (LDA). Moreover, each pure sweetener was quantified based on the emission intensities of selected sensor elements. The linear ranges for these sweeteners lie within 0.05–100 mM, but vary with the type of sweetener. The applicability of this array to real-life samples was demonstrated by applying it to various beverages, and the results showed that the sensor array possesses excellent discrimination power and reversibility. Figure Pattern recognition of ten sweeteners by LDA
Keywords: Chemiluminescence; Sensor array; Sweeteners; Discrimination; Linear discriminant analysis (LDA)

Toward the quantification of the 13CO2/12CO2 ratio in exhaled mouse breath with mid-infrared hollow waveguide gas sensors by Andreas Wilk; Felicia Seichter; Seong-Soo Kim; Erhan Tütüncü; Boris Mizaikoff; Josef A. Vogt; Ulrich Wachter; Peter Radermacher (397-404).
Mouse sepsis models are used to gain insight into the complex processes involved with patients suffering from glucose metabolism disorders. Measuring the expiratory release of 13CO2 after administering stable labeled 13C6-glucose enables assessment of the in vivo integrity and functionality of key metabolic processes. In the present study, we demonstrate that Fourier transform infrared spectroscopy operating in the mid-infrared spectral regime (2–20 μm) combined with hollow waveguide gas sensing modules simultaneously serving as a miniaturized gas cell and as a waveguide are capable of quantitatively monitoring 13CO2 enrichment levels in low volume mouse breath samples. Figure Mouse intensive care unit (MICU) with patient and IR signature of the time progression for 12CO2/13CO2 in exhaled mouse breath
Keywords: FTIR spectroscopy; Mid-infrared gas sensor; Hollow waveguide; Mouse sepsis model; Glucose/insulin metabolism disorder; 13CO2 enrichment

The aromatic peroxygenase from Marasmius rutola—a new enzyme for biosensor applications by Aysu Yarman; Glenn Gröbe; Bettina Neumann; Mathias Kinne; Nenad Gajovic-Eichelmann; Ulla Wollenberger; Martin Hofrichter; René Ullrich; Katrin Scheibner; Frieder W. Scheller (405-412).
The aromatic peroxygenase (APO; EC from the agraric basidomycete Marasmius rotula (MroAPO) immobilized at the chitosan-capped gold-nanoparticle-modified glassy carbon electrode displayed a pair of redox peaks with a midpoint potential of −278.5 mV vs. AgCl/AgCl (1 M KCl) for the Fe2+/Fe3+ redox couple of the heme-thiolate-containing protein. MroAPO oxidizes aromatic substrates such as aniline, p-aminophenol, hydroquinone, resorcinol, catechol, and paracetamol by means of hydrogen peroxide. The substrate spectrum overlaps with those of cytochrome P450s and plant peroxidases which are relevant in environmental analysis and drug monitoring. In M. rotula peroxygenase-based enzyme electrodes, the signal is generated by the reduction of electrode-active reaction products (e.g., p-benzoquinone and p-quinoneimine) with electro-enzymatic recycling of the analyte. In these enzyme electrodes, the signal reflects the conversion of all substrates thus representing an overall parameter in complex media. The performance of these sensors and their further development are discussed.
Keywords: Unspecific peroxygenase; Cytochrome P450; Biosensors; Phenolic substances

Probe accessibility effects on the performance of electrochemical biosensors employing DNA monolayers by Vanessa Biagiotti; Alessandro Porchetta; Sara Desiderati; Kevin W. Plaxco; Giuseppe Palleschi; Francesco Ricci (413-421).
Surface-confined DNA probes are increasingly used as recognition elements (or presentation scaffolds) for detection of proteins, enzymes, and other macromolecules. Here we demonstrate that the density of the DNA probe monolayer on the gold electrode is a crucial determinant of the final signalling of such devices. We do so using redox modified single-stranded and double-stranded DNA probes attached to the surface of a gold electrode and measuring the rate of digestion in the presence of a non-specific nuclease enzyme. We demonstrate that accessibility of DNA probes for binding to their macromolecular target is, as expected, improved at lower probe densities. However, with double-stranded DNA probes, even at the lowest densities investigated, a significant fraction of the immobilized probe is inaccessible to nuclease digestion. These results stress the importance of the accessibility issue and of probe density effects when DNA-based sensors are used for detection of macromolecular targets. Figure Here we demonstrate that the density of the DNA probe monolayer is a crucial determinant of the final signalling of DNA-bases sensors used for the detection of proteins, enzymes, and other macromolecules.
Keywords: Density; DNA sensor; SAM; Protein; Electrochemical

Use of the Gerchberg–Saxton algorithm in optimal coherent anti-Stokes Raman spectroscopy by D. S. Moore; S. D. McGrane; M. T. Greenfield; R. J. Scharff; R. E. Chalmers (423-428).
We are utilizing recent advances in ultrafast laser technology and recent discoveries in optimal shaping of laser pulses to significantly enhance the stand-off detection of explosives via control of molecular processes at the quantum level. Optimal dynamic detection of explosives is a method whereby the selectivity and sensitivity of any of a number of nonlinear spectroscopic methods are enhanced using optimal shaping of ultrafast laser pulses. We have recently investigated the Gerchberg–Saxton algorithm as a method to very quickly estimate the optimal spectral phase for a given analyte from its spontaneous Raman spectrum and the ultrafast laser pulse spectrum. Results for obtaining selective coherent anti-Stokes Raman spectra (CARS) for an analyte in a mixture, while suppressing the CARS signals from the other mixture components, are compared for the Gerchberg–Saxton method versus previously obtained results from closed-loop machine-learning optimization using evolutionary strategies. Figure Photo of an acousto-optic modulator based pulse shaper, with red lines denoting the laser beam path. Entering at the bottom left is the transform limited pulse (spectrogram inset shows wavelength versus time in a false color plot), and exiting at the bottom right is the the arbitrarily shaped pulse
Keywords: Coherent control; Gerchberg–Saxton algorithm; Coherent Raman; Nonlinear spectroscopy; Explosives detection

Determination of moisture content of single-wall carbon nanotubes by Ralph E. Sturgeon; Joseph W. Lam; Anthony Windust; Patricia Grinberg; Rolf Zeisler; Rabia Oflaz; Rick L. Paul; Brian E. Lang; Jeffrey A. Fagan; Benoit Simard; Christopher T. Kingston (429-438).
Several techniques were evaluated for the establishment of reliable water/moisture content of single-wall carbon nanotubes. Karl Fischer titration (KF) provides a direct measure of the water content and was used for benchmarking against results obtained by conventional oven drying, desiccation over anhydrous magnesium perchlorate as well as by thermogravimetry and prompt gamma-ray activation analysis. Agreement amongst results was satisfactory with the exception of thermogravimetry, although care must be taken with oven drying as it is possible to register mass gain after an initial moisture loss if prolonged drying time or elevated temperatures (120 °C) are used. Thermogravimetric data were precise but a bias was evident that could be accounted for by considering the non-selective loss of mass as volatile carbonaceous components. Simple drying over anhydrous magnesium perchlorate for a minimum period of 8–10 days is recommended if KF is not available for this measurement. Figure Drying over desiccants such as anhydrous Mg(ClO4)2 provides a convenient and inexpensive means of establishing a reproducible moisture content for SWCNT material
Keywords: Single-wall carbon nanotubes; Moisture determination; Oven drying; Desiccator drying; Karl Fischer titration; Prompt gamma-ray activation analysis

The aim of this work was speciation analysis of metabolites in feces samples collected within a clinical study during which a bromine-containing anti-tuberculosis drug (TMC207) was administered to patients with multi-drug resistant tuberculosis infection. Owing to slow elimination of the drug, no 14C label was used within this study. Quantification of the bromine species was accomplished using high performance liquid chromatography coupled to inductively coupled plasma–mass spectrometry (HPLC/ICP-MS) in combination with on-line isotope dilution (on-line ID), while structural elucidation of the species was performed using HPLC coupled to electrospray ionization–mass spectrometry. The ICP-MS-based method developed shows a good intra- and inter-day reproducibility (relative standard deviation = 3.5%, N = 9); the limit of detection (1.5 mg TMC207 L−1) is of the same order of magnitude as that for HPLC/radiodetection; the dynamic range of the method covers more than two orders of magnitude. Furthermore, the column recovery was demonstrated to be quantitative (recoveries between 90.6% and 99.5%). Based on the excellent figures of merit, the “cold” HPLC/ICP-MS approach could be deployed for the actual human in vivo metabolism study, such that exposure of the human volunteers to the 14C radiolabel was avoided. Figure HPLC/ICP-MS combined with on-line isotope dilution was applied for quantification of the metabolites of a bromine-containing anti-tuberculosis drug in feces extracts from a clinical trial (human in vivo study), thus avoiding the need for radiolabeling.
Keywords: HPLC/ICP-MS; Post-column on-line isotope dilution; Bromine speciation analysis; ESI-MS; Human in vivo study

In nonclinical drug development targeting the central nervous system (CNS), the quantitative determination of extracellular brain concentrations of neurotransmitters is a key challenge. In some CNS disorders, the monitoring of the modified profile of neurotransmitter release such as that of histamine may explain the mechanism of action of the drug candidate. Microdialysis is a commonly used method for sampling extracellular levels of neurotransmitters/drug candidates in small laboratory animals. Detection and quantification of extracellular levels of neurotransmitters remain an analytical and technical challenge owing to the low concentrations of neurotransmitters collected, the small microdialysis sample size, and the high amount of inorganic salts. A precolumn derivatization strategy prior to hydrophilic interaction liquid chromatography (HILIC)–tandem mass spectrometry analysis is proposed to quantify histamine release after administration of a CNS research compound. Derivatization using propionic anhydride dissolved in organic solvent combined with the HILIC approach effectively eliminated three time-consuming steps, organic layer transfer, dry down, and reconstitution, all of which are required by traditional reversed-phase liquid chromatography. The formation of propionylated amides, performed under mild conditions, required no further sample cleanup. After a dual microdialysis probe implantation into the prefrontal cortex (neurotransmitters) and in the inferior vena cava of rat (drug candidate), microdialysate fractions were collected every 15 min for 8 h and stored frozen at -20 °C until analysis. The method was validated using 10 μL microdialysate, achieving low limits of quantitation of 83.4 and 84.5 pg.mL-1 for histamine and 1-methylhistamine, respectively. These limits were suitable to assess kinetic release of neurotransmitters and are compatible with those obtained by microdialysis sampling. This method provided the required selectivity, sensitivity, accuracy, and precision to assess release kinetics of histamine and 1-methylhistamine in several hundred rat brain microdialysates after intravenous infusion of CNS drug candidates.
Keywords: Bioanalytical methods; Clinical/biomedical analysis; High-performance liquid chromatography; Mass spectrometry/inductively coupled plasma mass spectrometry

A ferrocene-based reagent for the conjugation and quantification of reactive metabolites by Sandra Jahn; Wiebke Lohmann; Susanne Bomke; Anne Baumann; Uwe Karst (461-471).
In the present study, a method for the analysis of reactive metabolites via liquid chromatography (LC) with inductively coupled plasma–mass spectrometry (MS) was developed. A ferrocenyl-modified glutathione (GSH) reagent, consisting of GSH and succinimidyl-3-ferrocenylpropionate, was synthesized. Derivatization of the tripeptide was performed at the N-terminus, leaving the nucleophilic thiol group vacant for the attack of electrophilic compounds. The potential of ferrocenylpropionate (FP)-GSH as a trapping agent for reactive metabolites was investigated using an electrochemical flow-through cell for metabolism simulation coupled online to a LC system with electrospray ionization mass spectrometric detection. The pharmaceuticals amodiaquine, an antimalarial agent, and clozapine, an antipsychotic compound, served as model substances. By proving the successful adduct formation between the reactive metabolite and ferrocene-labeled GSH, it could be shown that FP-GSH is an effective trapping agent which eases routine reversed-phase LC analyses. In contrast to GSH, which is usually used for the conjugation of reactive metabolites and where the resulting adducts often show no or only very little retention, FP-GSH facilitates the detection of the corresponding metabolite adducts due to higher retention times.
Keywords: Reactive metabolites; Ferrocene label; Electrochemistry; Mass spectrometry; Liquid chromatography; Oxidative metabolism

Development and certification of green tea-containing standard reference materials by L. C. Sander; M. Bedner; M. C. Tims; J. H. Yen; D. L. Duewer; B. Porter; S. J. Christopher; R. D. Day; S. E. Long; J. L. Molloy; K. E. Murphy; B. E. Lang; R. Lieberman; L. J. Wood; M. J. Payne; M. C. Roman; J. M. Betz; A. NguyenPho; K. E. Sharpless; S. A. Wise (473-487).
A suite of three green tea-containing Standard Reference Materials (SRMs) has been issued by the National Institute of Standards and Technology (NIST): SRM 3254 Camellia sinensis (Green Tea) Leaves, SRM 3255 Camellia sinensis (Green Tea) Extract, and SRM 3256 Green Tea-Containing Solid Oral Dosage Form. The materials are characterized for catechins, xanthine alkaloids, theanine, and toxic elements. As many as five methods were used in assigning certified and reference values to the constituents, with measurements carried out at NIST and at collaborating laboratories. The materials are intended for use in the development and validation of new analytical methods, and for use as control materials as a component in the support of claims of metrological traceability. Figure Green Tea - Camellia sinensis
Keywords: Green tea; Camellia sinensis ; Standard Reference Material; Catechins; Xanthines; Toxic elements; Theanine

The potential of a headspace device coupled to multi-capillary column-ion mobility spectrometry has been studied as a screening system to differentiate virgin olive oils (“lampante,” “virgin,” and “extra virgin” olive oil). The last two types are virgin olive oil samples of very similar characteristics, which were very difficult to distinguish with the existing analytical method. The procedure involves the direct introduction of the virgin olive oil sample into a vial, headspace generation, and automatic injection of the volatiles into a gas chromatograph-ion mobility spectrometer. The data obtained after the analysis by duplicate of 98 samples of three different categories of virgin olive oils, were preprocessed and submitted to a detailed chemometric treatment to classify the virgin olive oil samples according to their sensory quality. The same virgin olive oil samples were also analyzed by an expert’s panel to establish their category and use these data as reference values to check the potential of this new screening system. This comparison confirms the potential of the results presented here. The model was able to classify 97% of virgin olive oil samples in their corresponding group. Finally, the chemometric method was validated obtaining a percentage of prediction of 87%. These results provide promising perspectives for the use of ion mobility spectrometry to differentiate virgin olive oil samples according to their quality instead of using the classical analytical procedure.
Keywords: Headspace; Multi-capillary column; Ion mobility spectrometry; Virgin olive oil; Quality control

Development of single-chain variable fragment (scFv) antibodies against hapten benzo[a]pyrene: a binding study by Xaver Y. Z. Karsunke; Haifeng Wang; Ekkehard Weber; Michael D. McLean; Reinhard Niessner; J. Christopher Hall; Dietmar Knopp (499-507).
Due to its highly carcinogenic and mutagenic effect on humans, a maximum tolerable limit of 10 ng/L of benzo[a]pyrene (B[a]P) in drinking water was set by the European Commission (Council Directive 98/83/EC). Although several polyclonal and monoclonal antibodies (mAb) for the detection of B[a]P and other polycyclic aromatic hydrocarbons (PAH) have been developed by others, a traditional enzyme-linked immunosorbent assay (ELISA) with a limit of quantification of 10 ng/L for monitoring B[a]P has not been developed. With this in mind, several single-chain variable fragment (scFv) antibodies were created using existing mAbs against the extremely hydrophobic hapten B[a]P, and their heavy and light chains recombined to make unique variable light (VL) and heavy (VH) chain combinations. Their binding behaviour was investigated using microtiter plate ELISA and surface plasmon resonance techniques. Specifically, the coding sequences for VL and VH chains of 10 murine anti-B[a]P antibody producing hybridoma cell lines were isolated by degenerate oligonucleotide primer sets, cloned in phagemid pIT2 and transferred into Escherichia coli HB2151. To systematically investigate the interaction of the VL and VH domains, three high-affinity B[a]P-specific and one nonspecific clone were selected and recombined to build a set of 16 different VL and VH combinations. On the basis of our data, it was shown that the VH plays the major role for specific binding of B[a]P, whilst the VL can, in some cases, increase the final sensitivity of the assay by one order of magnitude. Furthermore, the sequence analysis of scFvs indicates that the complementarity determining region H3 plays a major role in affinity, whilst cross-reactivity to seven other PAHs demonstrates the importance of the VL in providing cross-reactivity. Figure Schematic drawing of a single-chain variable fragment antibody (scFv) for benzo[a]pyrene (B[a]P). It consists of the variable regions of light (VL) andheavy (VH) chains which are linked by a short peptide spacer.
Keywords: Benzo[a]pyrene; Recombinant antibodies; scFv; Binding affinity; Cross-reactivity; SPR

Determination of perfluorinated compounds in mollusks by matrix solid-phase dispersion and liquid chromatography–tandem mass spectrometry by Eugenia Villaverde-de-Sáa; José Benito Quintana; Rosario Rodil; Raúl Ferrero-Refojos; Elisa Rubí; Rafael Cela (509-518).
Perfluorinated compounds (PFCs) have been used for over 40 years in different commercial and industrial applications mainly as surfactants and surface protectors and have become an important class of marine emerging pollutants. This study presents the development and validation of a new analytical method to determine the simultaneous presence of eight PFCs in different kinds of mollusks using matrix solid-phase dispersion (MSPD) followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Simplicity of the analytical procedure, low volume of solvent and quantity of sample required, low global price, and integration of extraction and clean-up into a single step, are the most important advantages of the developed methodology. Solvent, solid support (dispersing agent), clean-up sorbent, and their amounts were optimized by means of an experimental design. In the final method, 0.5 g of sample are dispersed with 0.2 g of diatomaceous earth and transferred into a polypropylene syringe containing 4 g of silica as clean-up sorbent. Then, analytes are eluted with 20 mL of acetonitrile. The extract is finally concentrated to a final volume of 0.5 mL in methanol, avoiding extract dryness in order to prevent evaporation losses and injected in the LC-MS/MS. The combination of this MSPD protocol with LC-MS/MS afforded detection limits from 0.05 to 0.3 ng g−1. Also, a good linearity was established for the eight PFCs in the range from limit of quantification (LOQ) to 500 ng mL−1 with R 2 > 0.9917. The recovery of the method was studied with three types of spiked mollusk and was in the 64–126% range. Moreover, a mussel sample was spiked and aged for more than 1 month and analyzed by the developed method and a reference method, ion-pair extraction, for comparison, producing both methods statistically equal concentration values. The method was finally applied to the determination of PFCs in different kinds of mollusks revealing concentrations up to 8.3 ng g−1 for perfluoroundecanoic acid.
Keywords: Perfluorinated compounds (PFCs); Perfluorooctane sulfonate (PFOS); Perfluorooctanoic acid (PFOA); Matrix solid-phase dispersion (MSPD); Liquid chromatography–mass spectrometry (LC-MS); Mollusk; Mussel

A cost-effective and low solvent consumption method, based on the matrix solid-phase dispersion (MSPD) technique, for the determination of six benzotriazole UV absorbers in sediments is presented. Sieved samples (0.5 g) were first mixed in a mortar with a solid sorbent and then transferred to a polypropylene syringe containing a layer of clean-up co-sorbent. Analytes were eluted with a suitable solvent and further determined by gas chromatography with tandem mass spectrometry (GC-MS/MS). Under final conditions, diatomaceous earth and silica, deactivated to 10%, were used as inert dispersant and clean-up co-sorbent, respectively. Analytes were recovered using just 5 mL of dichloromethane, and this extract was concentrated and exchanged to 1 mL of isooctane. Further removal of co-extracted sulphur was achieved adding activated copper powder to final extracts, which were stored overnight, before injection in the GC-MS/MS system. The accuracy of the method was assessed with river and marine sediment samples showing different carbon contents and spiked at different concentrations in the range from 40 to 500 ng g−1. Recoveries varied between 78% and 110% with associated standard deviations below 14%. The limits of quantification of the method stayed between 3 and 15 ng g−1. Levels of target compounds in sediment samples ranged from not detected up to a maximum of 56 ng g−1 for Tinuvin 328.
Keywords: Benzotriazole UV absorbers; Sediment; Matrix solid-phase dispersion; Gas chromatography–mass spectrometry

Measurements in complex matrices like milk still present a challenge in biosensor development. This is especially important when using a label-free detection method or when measuring low analyte concentrations. The direct optical method reflectometric interference spectroscopy (RIfS) was used for investigating matrix effects in immunoassay development. Furthermore, approaches to reduce these effects have been established. As a model system, the hormone testosterone has been chosen because this immunoassay has been well characterized in buffer. In a first step, the immunoassay for the detection of testosterone in buffer was improved beyond former published results. Therefore, the sensor surface was optimized, resulting in a fivefold lower limit of detection (70.2 ng L−1) and limit of quantification (130.0 ng L−1). Additionally, the assay time could be reduced to 15 min. Consequently, we used this improved assay to investigate matrix effects of whole pasteurized bovine milk. To minimize these effects, the surface chemistry was adapted and a suitable evaluation method was established, reducing the effects of Tyndall scattering and nonspecific binding to the sensor surface. These improvements allow for very reliable quantitative measurements in milk. The assay developed required no sample pretreatment and allowed for the regeneration of the sensor surface so that calibration could be performed on one chip. The calibration in milk (3.5% fat) resulted in a limit of detection of 94.4 ng L−1 and a limit of quantification of 229.3 ng L−1. Furthermore, recovery rates between 70% and 120% could be obtained. Thus, for the first time, an analyte in the matrix milk was successfully quantified with RIfS at low concentrations.
Keywords: Reflectometric interference spectroscopy (RIfS); Milk analysis; Matrix effects; Testosterone; Label-free biosensor; Environmental analysis

Mass-spectrometric studies of new 6-nitroquipazines—serotonin transporter inhibitors by Janina Witowska-Jarosz; Małgorzata Jarończyk; Aleksander P. Mazurek; Ingebrigt Sylte; Andrzej J. Bojarski; Zdzisław Chilmonczyk; Maciej Jarosz (537-541).
Six synthesized 6-nitroquipazine derivatives were examined by electron ionization (EI) and electrospray ionization (ESI) mass spectrometry in positive and negative ion mode. The compounds exhibit high affinity for the serotonin transporter (SERT) and belong to a new class of SERT inhibitors. The EI mass spectra registered in negative ion mode showed prominent molecular ions for all the compounds studied. All EI mass spectra and all ESI mass spectra showed similar fragmentation pathways of molecular ions, but the pathways differed between EI and ESI. The differences were explained with the aid of theoretical evaluation of the stability of the respective radical ions (EI MS) and protonated ions (ESI MS). Figure Alkylnitroquipazines in SERT binding pocket superimposed on an exemplary MS fragmentation pattern
Keywords: Alkylnitroquipazines; Serotonin transporter inhibitors; Semiempirical geometry and charge distribution optimization; Electron ionization mass spectrometry; Electrospray ionization mass spectrometry

Solution-phase detection of dual microRNA biomarkers in serum by David Broyles; Kyle Cissell; Manoj Kumar; Sapna Deo (543-550).
A strategy for the simultaneous detection of multiple microRNA (miRNA) targets was developed utilizing fluorophore/quencher-labeled oligonucleotide probe sets. Two miRNA targets (miR-155 and miR-103), whose misregulation has afforded them status as putative biomarkers in certain types of cancer, were detected using our assay design. In the absence of target, the complementary fluorophore-probe and quencher-probe hybridize, resulting in a fluorescence resonance energy transfer-based quenching of the fluorescence signal. In the presence of unlabeled target, however, the antisense quencher-probe can hybridize with the target, resulting in increased fluorescence intensity as the quencher-probe is sequestered beyond the Förster radius of the fluorescent-probe. The assay design was tested in multiple matrices of buffer, cellular extract, and serum; and detection limits were found to be matrix-dependent, ranging from 0.34 to 8.89 pmol (3.4–59.3 nM) for miR-155 and 2.90–11.8 pmol (19.3–79.0 nM) for miR-103. Single, double, and triple nucleotide selectivity was also tested. Additionally, miR-155 concentrations were assessed in serum samples obtained directly from breast cancer patients without the need for RNA extraction. This assay is quantitative, possesses a low detection limit, can be applied in multiple complex matrices, and can obtain single-nucleotide selectivity. This method can be employed for the multiplex detection of solution-phase DNA or RNA targets and, more specifically, for the direct detection of serum miRNA biomarkers.
Keywords: miRNA; Detection; Fluorescence; FRET; Serum

Separation of inorganic anions on hydrophobic stationary phases in ion chromatography by Toyohide Takeuchi; Boran Jiang; Lee Wah Lim (551-555).
Inorganic anions were separated on hydrophobic stationary phases such as triacontyl-functionalized silica. Eluent conditions were examined in detail, and iodate, nitrate, iodide, and thiocyanate could be separated by using aqueous solutions. The effect of the eluent concentration on the retention of analyte anions was examined for a wide range of sodium sulfate concentrations of up to 1 M. The retention factor of hydrophobic anions decreased with increasing sodium sulfate concentration in the lower concentration region, while it increased with increasing sodium sulfate concentration in the higher concentration region. The addition of a small amount of an organic substance such as acetonitrile and tetraethylene glycol increased the retention of iodide and thiocyanate, while the addition of alcohols decreased their retention. Operating at lower temperature also increased the retention of analyte anions. It was expected that inorganic anions were retained on the stationary phase via hydrophobic interactions. The retention mechanism was discussed, considering the results obtained. Figa Retention of inorganic anions on a hydrophobic stationary phase
Keywords: Ion chromatography; Hydrophobic stationary phases; Retention behavior; Inorganic anions