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Analytical and Bioanalytical Chemistry (v.397, #8)

David D. Cunningham, Julie A. Stenken (Eds.): In vivo glucose sensing by Petr Herman (pp. 3159-3160).

Analytical tools for cell research by Edgar A. Arriaga; Simone König (pp. 3161-3162).

is a professor of chemistry, 2007–2008 Fesler–Lampert Chair in Aging Studies, Graduate Faculty of Biomedical Engineering, Gerontology, Molecular Biology, Biochemistry and Biophysics at the University of Minnesota, Minneapolis, MN (USA). He is a chartered member of the National Institutes of Health (NIH) Review Study Section Emerging Biotechnologies. His research program focuses on the development of bioanalytical methodologies to investigate tissue complexity, single cells and subcellular compartments. Applications of these methodologies include: (i) determining the subcellular fate and metabolism of xenobiotics, and (ii) investigating subcellular processes associated with the causes of aging. is a professor of biophysical chemistry (APL) at the Medical Faculty of the University of Münster. She has been head of the Proteomics Group of the Integrated Functional Genomics core unit of the Interdisciplinary Center for Clinical Research since completing her postdoctoral training with H.M. Fales at the NIH, Bethesda, USA (1995–1999). Her research interests involve methods and applications of biomolecular mass spectrometry. Recent studies include the real-time mass spectrometric interrogation of live insects at atmospheric pressure and label-free differential analysis.

Strategies for kinome profiling in cancer and potential clinical applications: chemical proteomics and array-based methods by Sander R. Piersma; Mariette Labots; Henk M. W. Verheul; Connie R. Jiménez (pp. 3163-3171).

Kinases are key enzymes involved in deregulated signal transduction associated with cancer development and progression. The advent of personalized medicine drives the development of new diagnostic tools for patient stratification and therapy selection Ginsburg and Willard (Transl Res 154:277-287, 2009). Since deregulation of kinase-mediated signal transduction is implied in tumorigenesis, the analysis of all kinases (the kinome) active in a particular tumor may yield tumor-specific information on aberrant cell signalling pathways. Tumor tissue kinase activity profiles may correlate with response to therapy and therefore may be used for future therapy selection. In this Trend paper we describe peptide array and mass spectrometry-based technologies and new developments for kinome profiling, and we present an outlook towards future implementation of therapy selection based on kinome profiling in clinical practice. Figure Protein kinases and interacting proteins are enriched by immobilized kinase inhibitor affinity capture, and their identity and phosphorylation sites are identified by mass spectrometry. Kinases that are differential between responders to therapy and non-responders can be used to design custom peptide substrate arrays to detect kinase-mediated aberrant signalling in cancer tissues. These custom peptide substrate arrays can be used, when validated, for therapy selection in the clinic.

Keywords: Kinase; Kinome profiling; Chemical proteomics; Peptide substrate array; Kinase inhibitor

Mammalian cell transfection: the present and the future by Tae Kyung Kim; James H. Eberwine (pp. 3173-3178).

Transfection is a powerful analytical tool enabling study of the function of genes and gene products in cells. The transfection methods are broadly classified into three groups; biological, chemical, and physical. These methods have advanced to make it possible to deliver nucleic acids to specific subcellular regions of cells by use of a precisely controlled laser-microcope system. The combination of point-directed transfection and mRNA transfection is a new way of studying the function of genes and gene products. However, each method has its own advantages and disadvantages so the optimum method depends on experimental design and objective.

Keywords: Transfection; Nucleic acid; Gene; Single cell

Interfacing droplet microfluidics with chemical separation for cellular analysis by Daniel T. Chiu (pp. 3179-3183).

This trends article discusses the interface between droplet microfluidics and micro-scale chemical separation. Droplet microfluidics has witnessed explosive growth over the past few years, but the use of droplets to facilitate chemical separation is still in its infancy. This article reviews the current state-of-the-art in this new area and provides an outlook on the role of this new technique in cellular analysis.

Keywords: Microfluidics; Droplets; Separation; CE; HPLC; Single cells

Mass spectral imaging and profiling of neuropeptides at the organ and cellular domains by Ruibing Chen; Lingjun Li (pp. 3185-3193).

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a rapid and sensitive analytical method that is well suited for determining molecular weights of peptides and proteins from complex samples. MALDI-MS can be used to profile the peptides and proteins from single-cell and small tissue samples without the need for extensive sample preparation. Furthermore, the recently developed MALDI imaging technique enables mapping of the spatial distribution of signaling molecules in tissue samples. Several examples of signaling molecule analysis at the single-cell and single-organ levels using MALDI-MS technology are highlighted followed by an outlook of future directions. Figure Overview of tissue-based mass spectrometric analysis strategies for neuropeptide discovery and distribution study. Direct MALDI analysis can be performed on single cells or small piece of tissues for neuropeptide profiling and novel neuropeptide discovery. MALDI imaging is powerful to study the spatial distribution of neuropeptides in larger and more complex organs, such as brain.

Keywords: Neuropeptides; Mass spectrometric imaging; MALDI-MS; Direct tissue profiling; Single-cell measurements

Interaction proteomics of synapse protein complexes by Ka Wan Li; Patricia Klemmer; August B. Smit (pp. 3195-3202).

The brain integrates complex types of information, and executes a wide range of physiological and behavioral processes. Trillions of tiny organelles, the synapses, are central to neuronal communication and information processing in the brain. Synaptic transmission involves an intricate network of synaptic proteins that forms the molecular machinery underlying transmitter release, activation, and modulation of transmitter receptors and signal transduction cascades. These processes are dynamically regulated and underlie neuroplasticity, crucial to learning and memory formation. In recent years, interaction proteomics has increasingly been used to elucidate the constituents of synaptic protein complexes. Unlike classic hypothesis-based assays, interaction proteomics detects both known and novel interactors without bias. In this trend article, we focus on the technical aspects of recent proteomics to identify synapse protein complexes, and the complementary methods used to verify the protein–protein interaction. Moreover, we discuss the experimental feasibility of performing global analysis of the synapse protein interactome.

Keywords: Brain; Synapse; Affinity isolation; Protein interactome

Novel antibody derivatives for proteome and high-content analysis by Katrin Schmidthals; Jonas Helma; Kourosh Zolghadr; Ulrich Rothbauer; Heinrich Leonhardt (pp. 3203-3208).

The understanding of cellular processes and their pathophysiological alterations requires comprehensive data on the abundance, distribution, modification, and interaction of all cellular components. On the one hand, artificially introduced fluorescent fusion proteins provide information about their distribution and dynamics in living cells but not about endogenous factors. On the other hand, antibodies can detect endogenous proteins, posttranslational modifications, and other cellular components but mostly in fixed and permeabilized cells. Here we highlight a new technology based on the antigen-binding domain of heavy-chain antibodies (V_HH) from Camelidae. These extremely stable V_HH domains can be produced in bacteria, coupled to matrices, and used for affinity purification and proteome studies. Alternatively, these V_HH domains can be fused with fluorescent proteins and expressed in living cells. These fluorescent antigen-binding proteins called “chromobodies” can be used to detect and trace proteins and other cellular components in vivo. Chromobodies can, in principle, detect any antigenic structure, including posttranslational modifications, and thereby dramatically expand the quality and quantity of information that can be gathered in high-content analysis. Depending on the epitope chosen, chromobodies can also be used to modulate protein function in living cells. Figure Detection of the nuclear lamina with lamin chromobody in living cells.

Keywords: Antibodies; Nanobodies; High-content analysis; Proteomics; Green fluorescent protein; Fluorescent proteins

Stepchild phosphohistidine: acid-labile phosphorylation becomes accessible by functional proteomics by Ulli Martin Hohenester; Katrin Ludwig; Josef Krieglstein; Simone König (pp. 3209-3212).

Bioanalytical techniques were preferentially developed for the investigation of phosphohydroxyamino acids in the past and there is a wealth of information on the detection of serine, threonine and tyrosine phosphorylation in functional proteomics. However, similarly important for protein regulation and signalling is the phosphorylation of other amino acids such as histidine, but its detection is hampered by the sensitivity to acid. Mass spectrometry in conjunction with chromatographic methods is allowing us to start to get a handle on phoshohistidine. ³²P-labelling and amino acid analysis for phosphorylation site determination is increasingly complemented by typical proteomic approaches based on reversed-phase peptide separation and gas-phase fragmentation. Chemical phosphorylation of peptides is a valuable tool, therefore, for the generation of analytical standards for use in method development. Figure RP-UPLC/Q-TOF MS/MS analysis of histidine-phosphorylated angiotensin II

Keywords: Phosphohistidine; Protein phosphorylation; Fragmentation; Mass spectrometry; Chromatography

Use of quantum dots in the development of assays for cancer biomarkers by Megan K. Wagner; Feng Li; Jingjing Li; Xing-Fang Li; X. Chris Le (pp. 3213-3224).

Biomarker assays may be useful for screening and diagnosis of cancer if a set of molecular markers can be quantified and statistically differentiated between cancerous cells and healthy cells. Markers of disease are often present at very low concentrations, so methods capable of low detection limits are required. Quantum dots (QDs) are nanoparticles that are emerging as promising probes for ultrasensitive detection of cancer biomarkers. QDs attached to antibodies, aptamers, oligonucleotides, or peptides can be used to target cancer markers. Their fluorescent properties have enabled QDs to be used as labels for in-vitro assays to quantify biomarkers, and they have been investigated as in-vivo imaging agents. QDs can be used as donors in assays involving fluorescence resonance energy transfer (FRET), or as acceptors in bioluminescence resonance energy transfer (BRET). The nanoparticles are also capable of electrochemical detection and are potentially useful for “lab-on-a-chip” applications. Recent developments in silicon QDs, non-blinking QDs, and QDs with reduced-size and controlled-valence further make these QDs bioanalytically attractive because of their low toxicity, biocompatibility, high quantum yields, and diverse surface modification flexibility. The potential of multiplexed sensing using QDs with different wavelengths of emission is promising for simultaneous detection of multiple biomarkers of disease. Figure Quantum dots have been conjugated to affinity probes to assay for cancer biomarkers including proteins, peptides, DNA, and whole cells

Keywords: Quantum dots; Nanoparticles; Tumor markers; Imaging; Biosensors; Fluorescence; FRET; BRET; Electrochemical; Multiplex; Aptamers; Cancer

Using aptamers to visualize and capture cancer cells by Ying Pu; Zhi Zhu; Huixia Liu; Jiani Zhang; Jun Liu; Weihong Tan (pp. 3225-3233).

Since diseased cells exist in exceedingly low concentration at the early stage of cancer, highly sensitive imaging and detection methods are required. By improving the methods for capturing and visualizing cancer cells, clinicians can diagnose metastatic relapse, stratify patients for therapeutic purposes, monitor response to drugs and therapies, and track tumor progression. Therefore, using advanced biotechnological and analytical methods combined with cell-SELEX (systematic evolution of ligands by exponential enrichment)-based aptamers, we improved the capture and visualization of diseased cells in a manner that is inexpensive, simple, sensitive, and fast. This multiplexed cancer detection platform therefore improves our control over a range of clinical exigencies, including cancer diagnosis, therapeutic modalities, and drug delivery systems.

Keywords: Aptamer; Cell-SELEX; Cancer cells; Imaging; Visualization; Nanomaterials; Clinical impact; Detection and enrichment

Single-cell electroporation by Manyan Wang; Owe Orwar; Jessica Olofsson; Stephen G. Weber (pp. 3235-3248).

Single-cell electroporation (SCEP) is a relatively new technique that has emerged in the last decade or so for single-cell studies. When a large enough electric field is applied to a single cell, transient nano-pores form in the cell membrane allowing molecules to be transported into and out of the cell. Unlike bulk electroporation, in which a homogenous electric field is applied to a suspension of cells, in SCEP an electric field is created locally near a single cell. Today, single-cell-level studies are at the frontier of biochemical research, and SCEP is a promising tool in such studies. In this review, we discuss pore formation based on theoretical and experimental approaches. Current SCEP techniques using microelectrodes, micropipettes, electrolyte-filled capillaries, and microfabricated devices are all thoroughly discussed for adherent and suspended cells. SCEP has been applied in in-vivo and in-vitro studies for delivery of cell-impermeant molecules such as drugs, DNA, and siRNA, and for morphological observations.

Keywords: Cell membrane; Electric field; Microelectrodes; Microfluidics; Transfection

Label-free cell separation and sorting in microfluidic systems by Daniel R. Gossett; Westbrook M. Weaver; Albert J. Mach; Soojung Claire Hur; Henry Tat Kwong Tse; Wonhee Lee; Hamed Amini; Dino Di Carlo (pp. 3249-3267).

Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible. Figure A wide range of microfluidic technologies have been developed to separate and sort cells by taking advantage of differences in their intrinsic biophysical properties

Keywords: Bioanalytical methods; Cell systems; Single cell analysis; Biochips; High-throughput screening; Microfluidics; Microfabrication; Separations; Instrumentation; Field-flow fractionation

Analytical approaches to investigate transmitter content and release from single secretory vesicles by Donna M. Omiatek; Ann-Sofie Cans; Michael L. Heien; Andrew G. Ewing (pp. 3269-3279).

The vesicle serves as the primary intracellular unit for the highly efficient storage and release of chemical messengers triggered during signaling processes in the nervous system. This review highlights conventional and emerging analytical methods that have used microscopy, electrochemistry, and spectroscopy to resolve the location, time course, and quantal content characteristics of neurotransmitter release. Particular focus is on the investigation of the synaptic vesicle and its involvement in the fundamental molecular mechanisms of cell communication.

Keywords: Vesicle; Exocytosis; Neurotransmitter; Carbon-fiber amperometry; Electrochemical cytometry; Capillary electrophoresis

Bioanalytical tools for single-cell study of exocytosis by Shencheng Ge; Secil Koseoglu; Christy L. Haynes (pp. 3281-3304).

Regulated exocytosis is a fundamental biological process used to deliver chemical messengers for cell-cell communication via membrane fusion and content secretion. A plethora of cell types employ this chemical-based communication to achieve crucial functions in many biological systems. Neurons in the brain and platelets in the circulatory system are representative examples utilizing exocytosis for neurotransmission and blood clotting. Single-cell studies of regulated exocytosis in the past several decades have greatly expanded our knowledge of this critical process, from vesicle/granule transport and docking at the early stages of exocytosis to membrane fusion and to eventual chemical messenger secretion. Herein, four main approaches that have been widely used to study single-cell exocytosis will be highlighted, including total internal reflection fluorescence microscopy, capillary electrophoresis, single-cell mass spectrometry, and microelectrochemistry. These techniques are arranged in the order following the route of a vesicle/granule destined for secretion. Within each section, the basic principles and experimental strategies are reviewed and representative examples are given revealing critical spatial, temporal, and chemical information of a secretory vesicle/granule at different stages of its lifetime. Lastly, an analytical chemist’s perspective on potential future developments in this exciting field is discussed.

Keywords: Single-cell study; Exocytosis; Total internal reflection fluorescence microscopy; Capillary electrophoresis; Mass spectrometry; Microelectrochemistry

Capillary array isoelectric focusing with laser-induced fluorescence detection: milli-pH unit resolution and yoctomole mass detection limits in a 32-channel system by Oluwatosin O. Dada; Lauren M. Ramsay; Jane A. Dickerson; Nathan Cermak; Rong Jiang; Cuiru Zhu; Norman J. Dovichi (pp. 3305-3310).

We report a multiplexed capillary electrophoresis system employing an array of 32 capillaries with a micromachined sheath-flow cuvette as the detection chamber. The sample streams were simultaneously excited with a 473-nm laser beam, and the fluorescence emission was imaged on a CCD camera with a pair of doublet achromat lens. The instrument produced mass detection limits of 380 ± 120 yoctomoles for fluorescein in zone electrophoresis. Capillary isoelectric focusing of fluorescent standards produced peaks with an average width of 0.0029 ± 0.0008 pH. Capillary coating stability limits the reproducibility of the analysis.

Keywords: Capillary electrophoresis/electrophoresis; Fluorescence/luminescence; Bioanalytical methods; Spectroscopy/instrumentation

Field-based ion generation from microscale emitters on natural and artificial objects for atmospheric pressure mass spectrometry by Alexander Pirkl; Klaus Dreisewerd; Joanne Y. Yew; Simone König (pp. 3311-3316).

Field-based ion generation is described for ambient mass spectrometry. The technique allows the analysis of endogenously expressed chemicals and exogenously applied compounds directly from the cuticle of live insects in real time. Cuticular hairs serve as electric field-enhancing structures and play a key role in ion generation. Artificial emitters such as graphite whiskers or sharp metal tips replicate this effect. Figure Fruit fly in front of the ion trap entrance capillary (see file Online_abstract_figure.jpg).

Keywords: Ions; Field-based ionization; Drosophila ; Mass spectrometry; Microstructured emitter

Probing of multidrug ABC membrane transporters of single living cells using single plasmonic nanoparticle optical probes by Kerry J. Lee; Lauren M. Browning; Tao Huang; Feng Ding; Prakash D. Nallathamby; Xiao-Hong Nancy Xu (pp. 3317-3328).

Currently, molecular mechanisms of multidrug ABC (ATP-binding cassette) membrane transporters remain elusive. In this study, we synthesized and characterized purified spherically shaped silver nanoparticles (Ag NPs) (11.8 ± 2.6 nm in diameter), which were stable (non-aggregation) in PBS buffer and inside single living cells. We used the size-dependent localized surface plasmon resonance (LSPR) spectra of single Ag NPs to determine their sizes and to probe the size-dependent transport kinetics of the ABC (BmrA, BmrA-EGFP) transporters in single living cells (Bacillus subtilis) in real time at nanometer resolution using dark-field optical microscopy and spectroscopy (DFOMS). The results show that the smaller NPs stayed longer inside the cells than larger NPs, suggesting size-dependent efflux kinetics of the membrane transporter. Notably, accumulation and efflux kinetics of intracellular NPs for single living cells depended upon the cellular expression level of BmrA, NP concentrations, and a pump inhibitor (25 μM, orthovanadate), suggesting that NPs are substrates of BmrA transporters and that passive diffusion driven by concentration gradients is the primary mechanism by which the NPs enter the cells. The accumulation and efflux kinetics of intracellular NPs for given cells are similar to those observed using a substrate (Hoechst dye) of BmrA, demonstrating that NPs are suitable probes for study of multidrug membrane transporters of single living cells in real-time. Unlike fluorescent probes, single Ag NPs exibit size-dependent LSPR spectra and superior photostability, enabling them to probe the size-dependent efflux kinetics of membrane transporters of single living cells in real-time for better understanding of multidrug resistance. Online abstract figure Probing of efflux functions of single multidrug transporters using single plasmonic nanoparticles

Keywords: ABC (BmrA) transporter; Single cell imaging; Multidrug resistance; Bacillus subtilis ; Single nanoparticle plasmonic optics; Ag nanoparticle

Multiplexed immunoassays for the analysis of breast cancer biopsies by Nicole Schneiderhan-Marra; Georg Sauer; Cornelia Kazmaier; Hsin-Yun Hsu; Karin Koretz; Helmut Deissler; Thomas O. Joos (pp. 3329-3338).

Within the last decade, protein microarray technology has been successfully used for the simultaneous quantification of target proteins from minimal amounts of samples in basic and applied proteome research. The robustness and appropriate sensitivity of these miniaturized assays have been demonstrated and thus the transfer to routine and high-throughput applications is now possible. In this study, multiplexed bead-based sandwich immunoassays were used to determine the concentrations of 54 protein analytes, including HER 2 and the estrogen receptor, from ultrasound-guided large-core needle biopsies (LCNBs) from breast cancer patients. Expression levels for HER 2, estrogen receptors and progesterone receptors were also assessed by immunohistochemical routine staining, performed in the clinic on corresponding biopsy samples. The high concordance of the data sets generated with the bead-based protein arrays and by conventional immunohistochemical assessment of HER 2 and the estrogen receptor expressed by breast cancer cells present in the biopsies was demonstrated.

Keywords: Breast cancer; Estrogen receptor; HER 2; Large-core needle biopsy; Protein array

Temporal resolution of protein–protein interactions in the live-cell plasma membrane by Julian Weghuber; Stefan Sunzenauer; Birgit Plochberger; Mario Brameshuber; Thomas Haselgrübler; Gerhard J. Schütz (pp. 3339-3347).

We have recently devised a method to quantify interactions between a membrane protein (“bait”) and a fluorophore-labeled protein (“prey”) directly in the live-cell plasma membrane (Schwarzenbacher et al. Nature Methods 5:1053–1060 2008). The idea is to seed cells on surfaces containing micro-patterned antibodies against the exoplasmic domain of the bait, and monitor the co-patterning of the fluorescent prey via fluorescence microscopy. Here, we characterized the time course of bait and prey micropattern formation upon seeding the cells onto the micro-biochip. Patterns were formed immediately after contact of the cells with the surface. Cells were able to migrate over the chip surface without affecting the micropattern contrast, which remained constant over hours. On single cells, bait contrast may be subject to fluctuations, indicating that the bait can be released from and recaptured on the micropatterns. We conclude that interaction studies can be performed at any time-point ranging from 5 min to several hours post seeding. Monitoring interactions with time opens up the possibility for new assays, which are briefly sketched in the discussion section.

Keywords: Protein–protein interactions; Temporal resolution; Micro-patterned surfaces; Atomic force microscopy; Fluorescence microscopy; Plasma membrane; Lipid rafts

Alterations in discrete glutamate receptor subunits in adult mouse dentate gyrus granule cells following perforant path transection by Stephen D. Ginsberg (pp. 3349-3358).

Custom-designed microarray analysis was utilized to evaluate expression levels of glutamate receptors (GluRs) and GluR-interacting protein genes within isolated dentate gyrus granule cells following axotomy of the principal input, the perforant path (PP). Dentate gyrus granule cells were evaluated by microdissection via laser capture microdissection, terminal continuation RNA amplification, and microarray analysis following unilateral PP transections at seven time points. Expression profiles garnered from granule cells on the side ipsilateral to PP transections were compared and contrasted with naive subjects and mice subjected to unilateral occipital cortex lesions. Selected microarray observations were validated by real-time quantitative PCR analysis. Postlesion time-dependent alterations in specific α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, kainate receptors, N-methyl-d-aspartate (NMDA) receptors, and GluR-interacting protein genes were found across the time course of the study, suggesting a neuroplasticity response associated with the transsynaptic granule cell alterations following axotomy of incoming PP terminals.

Keywords: AMPA receptor; Axotomy; Entorhinal cortex; Kainate receptor; Microarray; NMDA receptor; RNA amplification; Transsynaptic degeneration

Analysis of protein kinase A activity in insulin-secreting cells using a cell-penetrating protein substrate and capillary electrophoresis by Femina Rauf; Yiding Huang; Thusitha P. Muhandiramlage; Craig A. Aspinwall (pp. 3359-3367).

A cell-penetrating, fluorescent protein substrate was developed to monitor intracellular protein kinase A (PKA) activity in cells without the need for cellular transfection. The PKA substrate (PKAS) was prepared with a 6×histidine purification tag, an enhanced green fluorescent protein (EGFP) reporter, an HIV-TAT protein transduction domain for cellular translocation and a pentaphosphorylation motif specific for PKA. PKAS was expressed in Escherichia coli and purified by metal affinity chromatography. Incubation of PKAS in the extracellular media facilitated translocation into the intracellular milieu in HeLa cells, βTC-3 cells and pancreatic islets with minimal toxicity in a time and concentration dependent manner. Upon cellular loading, glucose-dependent phosphorylation of PKAS was observed in both βTC-3 cells and pancreatic islets via capillary zone electrophoresis. In pancreatic islets, maximal PKAS phosphorylation (83 ± 6%) was observed at 12 mM glucose, whereas maximal PKAS phosphorylation (86 ± 4%) in βTC-3 cells was observed at 3 mM glucose indicating a left-shifted glucose sensitivity. Increased PKAS phosphorylation was observed in the presence of PKA stimulators forskolin and 8-Br-cAMP (33% and 16%, respectively), with corresponding decreases in PKAS phosphorylation observed in the presence of PKA inhibitors staurosporine and H-89 (40% and 54%, respectively). Figure Analysis of PKA activity via chimeric fluorescent kinase substrate proteins

Keywords: Capillary electrophoresis; Protein kinase A; Cell-penetrating peptides; Fluorescent protein

Evaluating the effects of estradiol on endothelial nitric oxide stimulated by erythrocyte-derived ATP using a microfluidic approach by Suzanne Letourneau; Luiza Hernandez; Andrea N. Faris; Dana M. Spence (pp. 3369-3375).

Recently, estrogens have been reported to have protective effects against experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). Although the molecular mechanism for such a protective effect is currently incomplete, we hypothesized that estradiol may reduce the release of ATP from erythrocytes (ERYs), thereby lowering the production of nitric oxide (NO) by endothelial cells. Here, we report on the use of a microfluidic device to investigate the direct effects of the estrogen estradiol on endothelial cell nitric oxide production. In addition, the incorporation of a thin polycarbonate membrane into the device enabled the passage of ERYs through the device to determine indirect effects of estradiol on NO production that may be meditated by ERYs. When these ERYs were incubated with increasing concentrations of estradiol, the NO production from the endothelial cells was attenuated to a value that was only 59 ± 7% of ERYs in the absence of estradiol. This decrease in NO production coincides with reductions in ERY-derived ATP release in the presence of estradiol. Estradiol is typically reported to have NO-stimulating effects; however, such reports have employed in vitro experimental designs that include only a single cell type. To demonstrate the potential importance of this attenuation of ATP from ERYs, results from a small-scale study show that the ATP release obtained from healthy controls was 138 ± 21 nM (n = 18) while the release from the ERYs obtained from people with MS was 375 ± 51 nM (n = 11). The studies reported here involving multiple cells types (endothelial cells and ERYs) may lead to a reappraisal of the in vivo activities of estradiol.

Keywords: Bioanalytical methods; Biological samples; Biotechnological products; Cell systems/single-cell analysis; Microfluidics/microfabrication; Fluorescence/luminescence

Contact printing of arrayed microstructures by Wei Xu; Alicia M. Luikart; Christopher E. Sims; Nancy L. Allbritton (pp. 3377-3385).

A novel contact printing method utilizing a sacrificial layer of polyacrylic acid (PAA) was developed to selectively modify the upper surfaces of arrayed microstructures. The method was characterized by printing polystyrene onto SU-8 microstructures to create an improved substrate for a cell-based microarray platform. Experiments measuring cell growth on SU-8 arrays modified with polystyrene and fibronectin demonstrated improved growth of NIH 3T3 (93% vs. 38%), HeLa (97% vs. 77%), and HT1080 (76% vs. 20%) cells relative to that for the previously used coating method. In addition, use of the PAA sacrificial layer permitted the printing of functionalized polystyrene, carboxylate polystyrene nanospheres, and silica nanospheres onto the arrays in a facile manner. Finally, a high concentration of extracellular matrix materials (ECM), such as collagen (5 mg/mL) and gelatin (0.1%), was contact-printed onto the array structures using as little as 5 μL of the ECM reagent and without the formation of a continuous film bridge across the microstructures. Murine embryonic stem cells cultured on arrays printed with this gelatin hydrogel remained in an undifferentiated state indicating an adequate surface gelatin layer to maintain these cells over time.

Keywords: Contact printing; Microfabrication; SU-8; Cell culture; Micropallet

Temporal dynamics of receptor-induced apoptosis in an affinity microdevice by Randall D. Reif; Charmaine Aguas; Michelle M. Martinez; Dimitri Pappas (pp. 3387-3396).

The temporal dynamics of Fas-induced apoptosis is elucidated. Jurkat cells are captured on the affinity surface of a microdevice coated with anti-CD95, an antibody known to induce apoptosis in cells via the extrinsic (caspase 8) pathway. The timing of apoptosis induction is controlled by the binding of the cells to the surface. Once bound, the cells are continuously stained with the caspase probe, l-bisaspartic acid rhodamine 110 (D₂R), and the fluorescence of the cells was monitored for 6 h by light microscopy. This approach normalizes the temporal dynamics for each cell, as the binding event is also the start of apoptosis. In addition to providing the number of apoptotic cells over time, the fluorescence of individual cells can be monitored, providing information about the timing of caspase activity in each cell. The rate of caspase cleavage of D₂R in each cell is also measured and shows good agreement between the cells in a given population. The effects of the caspase inhibitor, z-VAD-FMK, on the timing of caspase activity are also investigated and are shown to dramatically slow the apoptotic process. In the future, other caspase probes could be used to provide additional information about the temporal dynamics of caspase activation. Additional techniques, such as fluorescence correlation spectroscopy, can be coupled to these methods to provide faster temporal response and help to elucidate the heterogeneity of the apoptosis process.

Keywords: Apoptosis; Caspase activity; Caspase inhibition; Cell affinity; CD95; Fluorescence correlation spectroscopy

Detection of heteroplasmy in individual mitochondrial particles by Bobby G. Poe III; Ciarán F. Duffy; Michael A. Greminger; Bradley J. Nelson; Edgar A. Arriaga (pp. 3397-3407).

Mitochondrial DNA (mtDNA) mutations have been associated with disease and aging. Since each cell has thousands of mtDNA copies, clustered into nucleoids of five to ten mtDNA molecules each, determining the effects of a given mtDNA mutation and their connection with disease phenotype is not straightforward. It has been postulated that heteroplasmy (coexistence of mutated and wild-type DNA) follows simple probability rules dictated by the random distribution of mtDNA molecules at the nucleoid level. This model has been used to explain how mutation levels correlate with the onset of disease phenotype and loss of cellular function. Nonetheless, experimental evidence of heteroplasmy at the nucleoid level is scarce. Here, we report a new method to determine heteroplasmy of individual mitochondrial particles containing one or more nucleoids. The method uses capillary cytometry with laser-induced fluorescence detection to detect individual mitochondrial particles stained with PicoGreen, which makes it possible to quantify the mtDNA copy number of each particle. After detection, one or more particles are collected into polymerase chain reaction (PCR) wells and then subjected to real-time multiplexed PCR amplification. This PCR strategy is suitable to obtain the relative abundance of mutated and wild-type mtDNA. The results obtained here indicate that individual mitochondrial particles and nucleoids contained within these particles are not heteroplasmic. The results presented here suggest that current models of mtDNA segregation and distribution (i.e., heteroplasmic nucleoids) need further consideration. Figure Setup for collection of individual mitochondrial particles into PCR vials after their laser-induced fluorescence detection (image in the background). Laser-induced fluorescence detection of individual mitochondria particles (upper right). Multiplex real time PCR of the collected sample reveals the presence of wild type and deleted mtDNA (i.e., heteroplasmy) when the traces cross the upper and lower thresholds, respectively (lower right).

Keywords: Mitochondria; Heteroplasmy; Mitochondrial DNA; Fluorescence; PCR; Deletion; Nucleoid

Introduction of a 20 kHz Nd:YVO4 laser into a hybrid quadrupole time-of-flight mass spectrometer for MALDI-MS imaging by Paul J. Trim; Marie-Claude Djidja; Sally J. Atkinson; Keith Oakes; Laura M. Cole; David M. G. Anderson; Philippa J. Hart; Simona Francese; Malcolm R. Clench (pp. 3409-3419).

A commercial hybrid quadrupole time–of–flight mass spectrometer has been modified for high-speed matrix-assisted laser desorption ionisation (MALDI) imaging using a short-pulse optical technology Nd:YVO₄ laser. The laser operating in frequency-tripled mode (λ = 355 nm) is capable of delivering 1.5-ns pulses of energy at up to 8 μJ at 5–10 kHz and 3 μJ at 20 kHz. Experiments to improve beam homogeneity and reduce laser speckle by mechanical vibration of the fibre-optic laser delivery system are reported along with data from trial and tissue imaging experiments using the modified instrument. The laser appeared to yield best results for MALDI-MS imaging experiments when operating at repetition rates 5–10 kHz. Combining this with raster imaging allowed images of rat brain sections to be recorded in 37 min. Similarly, images of the distribution of peptides in “on-tissue” digest experiments from tumour tissues were recorded in 1 h and 30 min rather than the 8-h acquisition time previously used. A brief investigation of targeted protein analysis/imaging by multiple reaction monitoring experiments “on-tissue” is reported. A total of 26 transitions were recorded over a 3-s cycle time and images of abundant proteins were successfully recorded.

Keywords: MALDI; Imaging; Raster; MRM; Vanadate

New ammunition for the proteomic reactor: strong anion exchange beads and multiple enzymes enhance protein identification and sequence coverage by Hu Zhou; Weimin Hou; Jean-Philippe Lambert; Daniel Figeys (pp. 3421-3430).

The enrichment and processing of proteomic samples prior to multi-dimensional chromatography remain a challenge in ‘gel-free’ proteomics. We previously reported the development of a microfluidic device called the “proteomic reactor” that relied on enriching proteins by using strong cation exchange (SCX) followed by trypsin digestion in an interstitial volume as little as 50 nL. Here, we report a novel proteomic reactor that is based on polymeric strong anion exchange (SAX) material to analyse proteomic samples. We also compare the performance of the SAX proteomic reactor to our previously reported SCX proteomic reactor for analysing complex yeast proteomes. Our results indicate that the SAX protein reactor preferentially identifies more acidic peptides and proteins compared to the SCX reactor. We show that the SAX and SCX reactors are complementary and that their combination increases the number of unique peptides and proteins identified by 50%. Furthermore, we show that the number of protein identified can be increased further by up to 40% using different proteolytic enzymes on the proteomic reactor.

Keywords: Proteomic reactor; Strong cation exchange; Strong anion exchange; Protein coverage; Microfluidic

Labelling of biopolymers: current status and future trends by Jörg Bettmer; Uwe Karst (pp. 3431-3432).

received his Ph.D. at the University of Münster, Germany (under the supervision of Professor Karl Cammann) and his habilitation in analytical chemistry at the University of Mainz, Germany (Professor Klaus G. Heumann). Since the summer of 2007 he has been working as a Ramón y Cajal researcher at the University of Oviedo, Spain (Professor Alfredo Sanz-Medel). His research interests are focussed on the development and application of MS-based hyphenated techniques in the fields of elemental speciation, metallomics, and quantitative proteomics and genomics. received his Ph.D. at the University of Münster, Germany in the group of Karl Cammann, and then moved to the University of Colorado in Boulder for a postdoctoral fellowship with Robert E. Sievers. After finishing his habilitation in Münster, he was appointed a Full Professor of Chemical Analysis at the University of Twente, The Netherlands. In 2005, he assumed his current position as Chair of Analytical Chemistry in Münster. His main research interests include hyphenated techniques, in particular focussing on pharmaceutical analysis, elemental speciation and metallomics.

Investigation of protein–protein interactions in living cells by chemical crosslinking and mass spectrometry by Andrea Sinz (pp. 3433-3440).

The identification of protein–protein interactions within their physiological environment is the key to understanding biological processes at the molecular level. However, the artificial nature of in vitro experiments, with their lack of other cellular components, may obstruct observations of specific cellular processes. In vivo analyses can provide information on the processes within a cell that might not be observed in vitro. Chemical crosslinking combined with mass spectrometric analysis of the covalently connected binding partners allows us to identify interacting proteins and to map their interface regions directly in the cell. In this paper, different in vivo crosslinking strategies for deriving information on protein–protein interactions in their physiological environment are described.

Keywords: Chemical crosslinking; Photoaffinity labeling; In vivo; Mass spectrometry

Oxidative protein labeling in mass-spectrometry-based proteomics by Julien Roeser; Rainer Bischoff; Andries P. Bruins; Hjalmar P. Permentier (pp. 3441-3455).

Oxidation of proteins and peptides is a common phenomenon, and can be employed as a labeling technique for mass-spectrometry-based proteomics. Nonspecific oxidative labeling methods can modify almost any amino acid residue in a protein or only surface-exposed regions. Specific agents may label reactive functional groups in amino acids, primarily cysteine, methionine, tyrosine, and tryptophan. Nonspecific radical intermediates (reactive oxygen, nitrogen, or halogen species) can be produced by chemical, photochemical, electrochemical, or enzymatic methods. More targeted oxidation can be achieved by chemical reagents but also by direct electrochemical oxidation, which opens the way to instrumental labeling methods. Oxidative labeling of amino acids in the context of liquid chromatography(LC)–mass spectrometry (MS) based proteomics allows for differential LC separation, improved MS ionization, and label-specific fragmentation and detection. Oxidation of proteins can create new reactive groups which are useful for secondary, more conventional derivatization reactions with, e.g., fluorescent labels. This review summarizes reactions of oxidizing agents with peptides and proteins, the corresponding methodologies and instrumentation, and the major, innovative applications of oxidative protein labeling described in selected literature from the last decade.

Keywords: Protein oxidation; Radical; Protein footprinting; Peptide cleavage

Glycan labeling strategies and their use in identification and quantification by L. R. Ruhaak; G. Zauner; C. Huhn; C. Bruggink; A. M. Deelder; M. Wuhrer (pp. 3457-3481).

Most methods for the analysis of oligosaccharides from biological sources require a glycan derivatization step: glycans may be derivatized to introduce a chromophore or fluorophore, facilitating detection after chromatographic or electrophoretic separation. Derivatization can also be applied to link charged or hydrophobic groups at the reducing end to enhance glycan separation and mass-spectrometric detection. Moreover, derivatization steps such as permethylation aim at stabilizing sialic acid residues, enhancing mass-spectrometric sensitivity, and supporting detailed structural characterization by (tandem) mass spectrometry. Finally, many glycan labels serve as a linker for oligosaccharide attachment to surfaces or carrier proteins, thereby allowing interaction studies with carbohydrate-binding proteins. In this review, various aspects of glycan labeling, separation, and detection strategies are discussed. Figure MALDI-FTICR-MS of 2AA-labeled total plasma N-glycans

Keywords: Capillary electrophoresis; Carbohydrate; Derivatization; Liquid chromatography; Mass spectrometry; Review

Organometallic derivatizing agents in bioanalysis by Susanne Bomke; Michael Sperling; Uwe Karst (pp. 3483-3494).

Over the last few decades, the development of several innovative hyphenated analytical techniques and their routine use in laboratories has led to new possibilities for the quantitative analysis of biomolecules. Today, the identification and quantification of biomolecules such as peptides and proteins are essential to answer important medical, pharmaceutical, and biological questions. To allow efficient detection and structure elucidation of biomolecules, several approaches including derivatization strategies were investigated and applied during recent years. This article summarizes the current approaches for labeling and presents the different types of organometallic derivatizing agents used as labels. Furthermore, their analytical potential with respect to quantification and structure elucidation for different applications in the field of bioanalysis is discussed.

Keywords: Mass spectrometry; ICP-MS; Organometals; Bioanalytical methods

Application of isotope dilution ICP–MS techniques to quantitative proteomics by Jörg Bettmer (pp. 3495-3502).

ICP–MS techniques based on isotope dilution analysis can be regarded as an emerging tool in quantitative protein analysis. Well-known concepts, for example species-specific and unspecific isotope dilution analysis, which promoted accurate and precise quantification in elemental speciation studies, have nowadays been transferred to the analysis of large biomolecules, e.g. proteins. Besides detection of heteroatom-containing proteins, the artificial introduction of metal-containing labels has attracted much attention and, as a consequence, ICP–MS-based isotope dilution techniques can serve as a valuable quantification tool. In particular, because isotope dilution ICP–MS techniques can enable absolute protein quantification, they can be regarded as an attractive technique in current and prospective proteomics. In this review, recent developments and applications will be highlighted and critically assessed.

Keywords: Isotope dilution analysis; Inductively coupled plasma-mass spectrometry; Hyphenated techniques; Quantitative proteomics; Protein labelling

Liquid chromatography with complementary electrospray and inductively coupled plasma mass spectrometric detection of ferrocene-labelled peptides and proteins by Susanne Bomke; Thorben Pfeifer; Björn Meermann; Wolfgang Buscher; Uwe Karst (pp. 3503-3513).

Succinimidylferrocenyl propionate (SFP) is introduced as labelling agent for amino functions in peptides and proteins. The resulting derivatives are characterised by considerably lower polarity compared with the native analytes and can thus be well separated by means of reversed phase liquid chromatography (RP-LC). The reaction products are characterised by electrospray ionisation mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS). A further advantage of the method is a simple and straightforward derivatisation protocol. Different basic and acidic model proteins as lysozyme, ß-lactoglobulin A and insulin were derivatised using SFP. Furthermore, the first dual-labelling strategy of thiol and amino groups with ferrocene-based reagents is presented. Whereas the amino groups were derivatised with SFP, the thiol groups were functionalised by reaction with ferrocenecarboxylic acid(2-maleimidoyl)ethylamide. Again, LC/ESI-MS is a suitable tool to characterise the modified peptides and proteins.

Keywords: Ferrocene-based reagents; Proteins; Derivatisation; Amino and thiol groups; LC/ESI-MS; LC/ICP-MS

Absolute quantification of superoxide dismutase (SOD) using species-specific isotope dilution analysis by Christian L. Deitrich; Sandra Braukmann; Andrea Raab; Caroline Munro; Barbara Pioselli; Eva M. Krupp; Jane E. Thomas-Oates; Jörg Feldmann (pp. 3515-3524).

Here we report for the first time the use of species-specific isotope dilution mass spectrometry for the absolute quantification of a metalloprotein using non-denaturing gel electrophoresis laser ablation inductively coupled plasma mass spectrometry (GE-LA-ICP-MS). The concept utilises the intrinsic metals of the metalloprotein for labelling of the isotopically labelled spike (⁶⁵Cu, ⁶⁸Zn SOD). The stability of the metal–protein complex under non-denaturing conditions during 1-D PAGE was confirmed and the performance of the method evaluated. Between 4 and 64 μg, SOD was quantified with a recovery rate between 82% and 110% in a standard. The use of the isotopically enriched SOD was utilised to identify the extent of orthogonal diffusion in 1-D gel electrophoresis. Orthogonal diffusion of natural and isotopically enriched SOD in the gel can interfere with the correct determination of the isotope ratios. The matrix effect of a cytosolic liver extract on the non-covalently bound copper and zinc in SOD was evaluated and no significant metal loss from the SOD spike was observed. This study represents the first step necessary for establishing and evaluating the use of a species-specific isotope dilution approach for the absolute quantification of SOD in real samples based on the combination of gel electrophoresis and LA-ICP-MS.

Keywords: Isotope dilution mass spectrometry; ICP-MS; Protein quantification; Superoxide dismutase; Gel electrophoresis; Laser ablation; Speciation analysis

Investigation of the interaction of Mercurochrome® constituents with proteins using liquid chromatography/mass spectrometry by Andrea Wilken; Rasmus Janzen; Michael Holtkamp; Sascha Nowak; Michael Sperling; Martin Vogel; Uwe Karst (pp. 3525-3532).

The interaction of Mercurochrome®, a medical preparation based on the mercuryorganic compound merbromin, with free thiols in low molecular weight peptides and in proteins has been investigated by means of liquid chromatography (LC) and electrospray mass spectrometry (ESI-MS). β-Lactoglobulin A (β-LGA) from bovine milk (18.4 kDa) has been used as the model protein. It was found that, in contrast to assumptions in literature, the commercial product itself is a heterogeneous mixture of moderate chemical stability, which may contain precipitated Hg salts depending on storage time and conditions. Further variability results from different degrees of bromination of the fluorescein backbone of the compound. The formation of mercury compound–protein adducts was detected. The peptide sequence T13 containing a free thiol residue was identified as the binding site for mercury species after tryptic digestion of β-lactoglobulin A. While fresh Mercurochrome® tends to the formation of a Hg(II)-β-LGA adducts due to excess Hg²⁺ in solution, investigations after precipitation of Hg salts yield Hg(merbromin)(β-LGA) as the major product.

Keywords: Merbromin; Mercurochrome®; Mercury; Proteins; Thiols; Liquid chromatography; Mass spectrometry

Microarray-based amplification and detection of RNA by nucleic acid sequence based amplification by Andreas Mader; Ulrike Riehle; Thomas Brandstetter; Elmar Stickeler; Axel zur Hausen; Jürgen Rühe (pp. 3533-3541).

Nucleic acid sequence based amplification (NASBA) is a versatile in vitro nucleic acid amplification method. In this work, RNA amplification and labeling by NASBA and microarray analysis are combined in a one-step process. The NASBA reaction is performed in direct contact with capture probes. These probes are bound to surface-attached hydrogel spots generated at the chip surfaces by using a simple printing and UV irradiation process. Five gene expression and SNP parameters with known relevance in breast cancer diagnostics were chosen to demonstrate that multiplex NASBA-on-microarray analysis is possible. A minimum amount of 10 pg of total RNA was shown to be sufficient for the detection of the reference parameter RPS18, which demonstrates that the detection limit of the microarray-based NASBA assays theoretically allows single-cell assays to be performed. Figure The nucleic acid sequence based amplification (NASBA) reaction taking place on top of a microarray. a: During the NASBA process, biotinylated antisense RNA is produced. b: Hybridization of these amplicons with their immobilized capture probes occurs during amplification. Capture probes are immobilized within spots of a surface-attached hydrogel, arranged in a microarray. Each spot potentially represents a different capture probe. ds cDNA doubled-stranded complementary DNA, PMMA polymethylmethacrylate

Keywords: Nucleic acid sequence based amplification; Microarray; On-chip; Isothermal; RNA amplification

Analysis of intact ladderane phospholipids, originating from viable anammox bacteria, using RP-LC-ESI-MS by Ingela Lanekoff; Roger Karlsson (pp. 3543-3551).

Since the discovery of the anaerobic ammonium oxidizing (anammox) bacteria, many attempts have been made in order to identify these environmentally important bacteria in natural environments. Anammox bacteria contain a unique class of lipids, called ladderane lipids and here we present a novel method to detect viable anammox bacteria in sediments and waste water treatment plants based on the use of a ladderane lipid biomarker. Intact ladderane phosphatidylcholine (PC) lipids are analyzed using reversed-phase liquid chromatography–electrospray ionization–mass spectrometry. Following extraction from the complex sediment matrix, reversed-phase LC is used to separate ladderane PC lipids based on their tail group hydrophobicity as well as their ether or ester link to the glycerol backbone in the sn-2 position. We investigate the presence of intact ladderane lipids in natural sediments displaying anammox activity and illustrate the use of a specific intact membrane forming PC lipid as a biomarker for viable anammox bacterial cells. The presented method can be used to elucidate the whereabouts of viable anammox bacteria, subsequently enabling an estimation of anammox activity. This will greatly increase the knowledge of anammox bacteria and their importance in the global nitrogen cycle.

Keywords: Ladderane lipids; LCMS; Biomarker; Anammox bacteria; Nitrogen cycle

Label-free amperometric immunobiosensor based on a gold colloid and Prussian blue nanocomposite film modified carbon ionic liquid electrode by Ke-Jing Huang; De-Jun Niu; Jun-Yong Sun; Xiao-Li Zhu; Jun-Jie Zhu (pp. 3553-3561).

A novel experimental methodology based on a Prussian blue (PB) and gold nanoparticles (AuNPs) modified carbon ionic liquid electrode (CILE) was developed for use in a label-free amperometric immunosensor for the sensitive detection of human immunoglobulin G (HIgG) as a model protein. The CILE was fabricated by using the ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate as binder. Controllable electrodeposition of PB on the surface of the CILE and coating with 3-aminopropyl triethylene silane (APS) formed a film with high electronic catalytic activity and large surface area for the assembly of AuNPs and further immobilization of HIgG antibody. The electrochemistry of the formed nanocomposite biofilm was investigated by electrochemical techniques including cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy. The HIgG concentration was measured through the decrease of amperometric responses in the corresponding specific binding of antigen and antibody. The decreased differential pulse voltammetric values were proportional to the HIgG concentration in two ranges, 0.05–1.25 ng mL⁻¹ and 1.25–40 ng mL⁻¹, with a detection limit of 0.001 ng mL⁻¹ (S/N = 3). This electrochemical immunoassay combined the specificity of the immunological reaction with the sensitivity of the AuNPs, ionic liquid, and PB amplified electrochemical detection and would therefore be valuable for clinical immunoassays. Figure Cyclic voltammograms of the immunosensor in pH 7.0 phosphate-buffered solution containing 0.1 M KCl at the different scan rate of (from inner to outer) 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, and 800 mVs⁻¹. The inset shows the linear relationship between the peak currents and the square root of scan rate.

Keywords: Amperometric immunosensor; Prussian blue; Au nanoparticles; Human immunoglobulin G; Carbon ionic liquid electrode

Accurate quantification of mercapturic acids of styrene (PHEMAs) in human urine with direct sample injection using automated column-switching high-performance liquid chromatography coupled with tandem mass spectrometry by M. Reska; E. Ochsmann; T. Kraus; T. Schettgen (pp. 3563-3574).

Styrene is one of the most important industrial chemicals, with an enormously high production volume worldwide. The urinary mercapturic acids of its metabolite styrene-7,8-oxide, namely N-acetyl-S-(2-hydroxy-1-phenylethyl)-l-cysteine (PHEMA 1) and N-acetyl-S-(2-hydroxy-2-phenylethyl)-l-cysteine (PHEMA 2), are specific biomarkers for the determination of individual internal exposure to this highly reactive intermediate of styrene. We have developed and validated a fast, specific and very sensitive method for the accurate determination of the sum of phenylhydroxyethyl mercapturic acids (PHEMAs) in human urine with an automated multidimensional liquid chromatography–tandem mass spectrometry method using ¹³C₆-labelled PHEMAs as internal standards. Analytes were stripped from the urinary matrix by online extraction on a restricted access material, transferred to the analytical column and subsequently determined by tandem mass spectrometry. The limit of quantification (LOQ) for the sum of PHEMAs was 0.3 μg/L urine and allowed us to quantify the background exposure of the (smoking) general population. Precision within series and between series ranged from 1.5 to 6.8% at three concentrations ranging from 3 to 30 μg/L urine; the mean accuracy was between 104 and 110%. We applied the method to spot urine samples from 40 subjects of the general population with no known occupational exposure to styrene. The median levels (range) for the sum of PHEMAs in urine of non-smokers (n = 22) were less than 0.3 μg/L (less than 0.3 to 1.1 μg/L), whereas in urine of smokers (n = 18), the median levels were 0.46 μg/L (less than 0.3 to 2.8 μg/L). Smokers showed a significantly higher excretion of the sum of PHEMAs (p = 0.02). Owing to its automation and high sensitivity, our method is well suited for application in occupational or environmental studies.

Keywords: Biological monitoring; General population; Styrene oxide; Urine; Mercapturic acid

Study of ε-caprolactone polymerization by NIR spectroscopy by Marcelo Blanco; M. Jesús Sánchez; Manel Alcalà (pp. 3575-3579).

Near-infrared (NIR) spectroscopy is proposed for the in-line quantitative and kinetic study of the polymerization of ε-caprolactone and eventually to facilitate real-time control of the manufacturing process. Spectra were acquired with a fibre-optic probe operating in transflectance mode immersed in the reactor. The NIR data acquired were processed using a multivariate curve resolution alternating least squares (MCR-ALS) algorithm. The proposed method allows calculation of the concentration and spectral profiles of the species involved in the reaction. The key point of this method is the lack of reference concentrations needed to perform the MCR-ALS method. The use of an extended spectral matrix using both process and pure analyte spectra solves the rank deficiency. The concentration profiles obtained were used to calculate a kinetic fitting of the reaction, but the method was improved by applying kinetic constraints (hard modelling). The rate constants of batches at different temperatures and the energy of activation for this reaction were calculated. Whenever possible, the hard modelling combined with the MCR-ALS method improves the fit of the experimental data: the results show good correlation between the NIR and reference data and allow the collection of high-quality kinetic information on the reaction (rate constants and energy of activation).

Keywords: Near-infrared spectroscopy; Process analysis; Chemometrics; Multivariate curve resolution; Kinetics

Direct detection and discrimination of double-stranded oligonucleotide corresponding to hepatitis C virus genotype 3a using an electrochemical DNA biosensor based on peptide nucleic acid and double-stranded DNA hybridization by M. H. Pournaghi-Azar; F. Ahour; M. S. Hejazi (pp. 3581-3587).

Development of an electrochemical DNA biosensor for the direct detection and discrimination of double-stranded oligonucleotide (dsDNA) corresponding to hepatitis C virus genotype 3a, without its denaturation, using a gold electrode is described. The electrochemical DNA sensor relies on the modification of the gold electrode with 6-mercapto-1-hexanol and a self-assembled monolayer of 14-mer peptide nucleic acid probe, related to the hepatitis C virus genotype 3a core/E1 region. The increase of differential pulse voltammetric responses of methylene blue, upon hybridization of the self-assembled probe with the target ds-DNA to form a triplex is the principle behind the detection and discrimination. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the developed DNA sensor responds selectively to the ds-DNA target. Diagnostic performance of the biosensor is described and the detection limit was found to be 1.8 × 10⁻¹² M in phosphate buffer solution, pH 7.0. The relative standard deviation of measurements of 100 pM of target ds-DNA performed with three independent probe-modified electrodes was 3.1%, indicating a remarkable reproducibility of the detection method. Figure Development of an electrochemical DNA biosensor for the direct detection and discrimination of double-stranded oligonucleotide (dsDNA) corresponding to hepatitis C virus genotype 3a without its denaturation using a gold electrode is described. The Electrochemical DNA sensor relies on the modification of gold electrode with 6-mercapto-1-hexanol and self-assembled monolayer of 14-mer peptide nucleic acid probe, related to the hepatitis C virus genotype 3a core/E1 region. The increase of differential pulse voltammetric responses of methylene blue, upon hybridization of the self-assembled probe with the target ds-DNA to form a triplex is the principal of the detection and discrimination. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the ds-DNA target. Diagnostic performance of the biosensor is described and the detection limit was found to be 1.84 × 10⁻¹² M, in phosphate buffer solution, pH 7.0. The relative standard deviation over three independently probe modified electrodes measured at 100 pM of target ds-DNA was 3.1 % indicating a remarkable reproducibility of the detection method.

Keywords: DNA biosensor; Hepatitis C; PNA probe; Triplex formation; Gold electrode

Determination of vanadium(V) with CdTe quantum dots as fluorescent probes by Ming Hou; Jia Na (pp. 3589-3593).

CdTe quantum dots (QDs) were modified with thioglycolic acid (TGA) and synthesized in aqueous medium. The optimum fluorescence intensity was found to be at pH 6.24 with a CdTe QDs concentration of 4.96 × 10⁻⁷ mol L⁻¹. The quenched fluorescence intensity of CdTe QDs is linearly proportional to V(V) concentration from 10 to 200 ng mL⁻¹ with correlation coefficient R = 0.9985. The limit of detection for V(V) was 2.07 ng mL⁻¹. The proposed method was successfully applied to the analysis of trace amounts of V(V) in water samples with recovery of 96.5–101.8%, and the results were in good agreement with those of electrothermal atomic absorption spectrometry. Figure Fluorescence spectra of CdTe-V(V) system at different CV(V): a, 0 μg/mL; b, 0.01 μg/mL; c, 0.03 μg/mL; d,0.05 μg/mL; e, 0.1 μg/mL

Keywords: CdTe quantum dots; V(V) ions; Fluorescence; Quenching; Water

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Analytical and Bioanalytical Chemistry (v.397, #8)