Analytical and Bioanalytical Chemistry (v.406, #14)
Educating tomorrow’s chemists by Reiner Salzer (3251-3255).
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.Currently, he is Chairman of the ECTN Label Committee for the quality labels Chemistry Eurobachelor®, Chemistry Euromaster®, and Chemistry Doctorate Eurolabel®.
11th Dresdner Sensor Symposium—a conference on sensors, measurement techniques, and medical engineering by Julia Widmaier; Kathrin Freudenberger; Sabrina Rau; Urs Hilbig (3257-3258).
IMA 2013 - The 8th International Conference on Instrumental Methods of Analysis - Modern Trends and Applications by Maria Ochsenkühn-Petropoulou (3259-3260).
Multiplex platforms in diagnostics and bioanalytics by Günter Peine; Günther Proll (3261-3262).
is a biologist and Head of the Center for Molecular Diagnostics and Bioanalysis Berlin-Brandenburg (ZMDB), which supports biomarker discovery, assay development, and clinical validations in coopertion with the Charité Universitätsmedizin Berlin, scientific institutions and industry. He is member of the board of the “European Diagnostic Clusters Alliance – EDCA”, which is comprisesd of about 200 scientific institutions and 600 companies in the field of diagnostics in the United Kingdom, Belgium, France, Spain, Sweden, Finland, and Poland. On behalf of the governments of the states of Berlin and Brandenburg, he is responsible for the development of biotechnology and pharma in the German capital region. is a biosensor scientist at the Institute for Physical and Theoretical Chemistry of the University of Tuebingen. His work is focused on the characterization and development of optical transduction technologies, spatially resolved surface chemistry on diverse substrates, and multianalyte/multiplex assay development for molecular diagnostics and environmental applications. He has published 30 original articles and three book chapters. He is a member of the board of the chemo- and biosensors working group of the GDCh (German Chemical Society).
Cellphone-based devices for bioanalytical sciences by Sandeep Kumar Vashist; Onur Mudanyali; E. Marion Schneider; Roland Zengerle; Aydogan Ozcan (3263-3277).
During the last decade, there has been a rapidly growing trend toward the use of cellphone-based devices (CBDs) in bioanalytical sciences. For example, they have been used for digital microscopy, cytometry, read-out of immunoassays and lateral flow tests, electrochemical and surface plasmon resonance based bio-sensing, colorimetric detection and healthcare monitoring, among others. Cellphone can be considered as one of the most prospective devices for the development of next-generation point-of-care (POC) diagnostics platforms, enabling mobile healthcare delivery and personalized medicine. With more than 6.5 billion cellphone subscribers worldwide and approximately 1.6 billion new devices being sold each year, cellphone technology is also creating new business and research opportunities. Many cellphone-based devices, such as those targeted for diabetic management, weight management, monitoring of blood pressure and pulse rate, have already become commercially-available in recent years. In addition to such monitoring platforms, several other CBDs are also being introduced, targeting e.g., microscopic imaging and sensing applications for medical diagnostics using novel computational algorithms and components already embedded on cellphones. This report aims to review these recent developments in CBDs for bioanalytical sciences along with some of the challenges involved and the future opportunities. Figure The universal Rapid Diagnostic Test (RDT) reader developed at UCLA. It can read various lateral flow assays for point-of-care and telemedicine applications
Keywords: Cellphone; Bioanalytical sciences; Diagnostics; Point-of-care; Digital health
High-throughput, multiparameter analysis of single cells by Thomas Haselgrübler; Michaela Haider; Bozhi Ji; Kata Juhasz; Alois Sonnleitner; Zsolt Balogi; Jan Hesse (3279-3296).
Heterogeneity of cell populations in various biological systems has been widely recognized, and the highly heterogeneous nature of cancer cells has been emerging with clinical relevance. Single-cell analysis using a combination of high-throughput and multiparameter approaches is capable of reflecting cell-to-cell variability, and at the same time of unraveling the complexity and interdependence of cellular processes in the individual cells of a heterogeneous population. In this review, analytical methods and microfluidic tools commonly used for high-throughput, multiparameter single-cell analysis of DNA, RNA, and proteins are discussed. Applications and limitations of currently available technologies for cancer research and diagnostics are reviewed in the light of the ultimate goal to establish clinically applicable assays. Figure ᅟ
Keywords: Single-cell analysis; Population heterogeneity; Microfluidics; High-throughput screening; Genomics/proteomics; Nucleic acids (DNA/RNA)
Developing new materials for paper-based diagnostics using electrospun nanofibers by S. J. Reinholt; A. Sonnenfeldt; A. Naik; M. W. Frey; A. J. Baeumner (3297-3304).
The use of electrospun nanofibers as functional material in paper-based lateral flow assays (LFAs) was studied. Specific chemical features of the nanofibers were achieved by doping the base polymer, poly(lactic acid) (PLA), with poly(ethylene glycol) (PEG) and polystyrene8K-block-poly(ethylene-ran-butylene)25K-block-polyisoprene10K-Brij76 (K3-Brij76) (KB). The LFAs were assembled such that the sample flowed through the nanofiber mat via capillary action. Initial investigations focused on the sustainable spinning and assembly of different polymer structures to allow the LFA format. Here, it was found that the base polymer poly(vinyl alcohol) (PVA), which was shown to function well in microfluidic biosensors, did not work in the LFA format. In contrast, PLA-based nanofibers enabled easy assembly. Three relevant features were chosen to study nanofiber-based functionalities in the LFA format: adsorption of antibodies, quantification of results, and nonspecific binding. In particular, streptavidin-conjugated sulforhodamine B (SRB)-encapsulating liposomes were captured by anti-streptavidin antibodies adsorbed on the nanofibers. Varying the functional polymer concentration within the PLA base enabled the creation of distinct capture zones. Also, a sandwich assay for the detection of Escherichia coli O157:H7 was developed using anti-E. coli antibodies as capture and reporter species with horseradish peroxidase for signal generation. A dose–response curve for E. coli with a detection limit of 1.9 × 104 cells was achieved. Finally, functional polymers were used to demonstrate that nonspecific binding could be eliminated using antifouling block copolymers. The enhancement of paper-based devices using functionalized nanofibers provides the opportunity to develop a broad spectrum of sensitive and specific bioassays with significant advantages over their traditional counterparts. Figure Schematic of LFA format and single-step binding assay. A 1.75 × 5-mm nanofiber mat was placed directly on a backing card 4.5 mm in width, and a 1 × 20-cm absorbent pad was placed on the backing card overlapping the nanofiber mat by approximately 2 mm (a). The LFAs ran vertically in glass culture tubes. In the E. coli sandwich assay, E. coli (green) flowed through the anti-E. coli-modified nanofiber mat, followed by horseradish peroxidase (HRP)-conjugated (pink) anti-E. coli. When E. coli is present, a colorimetric signal results upon addition of HRP substrate (b), and when no E. coli is present, the HRP flows through the nanofiber mat and no signal is observed (c)
Keywords: Electrospun nanofibers; LFA; Poly(lactic acid); Point-of-care; Immunoassay; Liposomes
Development of a new parallelized, optical biosensor platform for label-free detection of autoimmunity-related antibodies by Oliver Bleher; Aline Schindler; Meng-Xin Yin; Andrew B. Holmes; Peter B. Luppa; Günter Gauglitz; Günther Proll (3305-3314).
Autoimmune diseases are characterized by the presence of autoantibodies in serum of affected patients. The heterogeneity of autoimmune relevant antigens creates a variety of different antibodies, which requires a simultaneous detection mode. For this reason, we developed a tool for parallelized, label-free, optical detection that accomplishes the characterization of multiple antigen–antibody interactions within a single measurement on a timescale of minutes. Using 11-aminoundecyltrimethoxysilane, we were able to immobilize proteinogenic antigens as well as an amino-functionalized cardiolipin on a glass surface. Assay conditions were optimized for serum measurements with a single spot antigen chip on a single spot 1-λ detection system. Minimized background signal allows a differentiation between patients and healthy controls with a good sensitivity and specificity. Applying polarized imaging reflectometric interference spectroscopy, we evaluated samples from three APS patients and three control subjects for this proof-of-principle and already obtained good results for β2-glycoprotein I and cardiolipin.
Keywords: Label-free detection; Optical sensors; Antiphospholipid syndrome; β2-Glycoprotein I; Reflectometric interference spectroscopy; Microarray
Nanoparticle-encapsulated vis- and NIR-emissive fluorophores with different fluorescence decay kinetics for lifetime multiplexing by Katrin Hoffmann; Thomas Behnke; Markus Grabolle; Ute Resch-Genger (3315-3322).
Bioanalytical, clinical, and security applications increasingly require simple, efficient, and versatile strategies to measure an ever increasing number of analytes or events in parallel in a broad variety of detection formats as well as in conjunction with chromatographic separation techniques or flow cytometry. An attractive alternative to common optical multiplexing and encoding methods utilizing spectral multiplexing/color encoding and intensity encoding is lifetime multiplexing, which relies on the discrimination between different fluorescent reporters based on their fluorescence decay kinetics. Here, we propose a platform of surface-functionalizable polymeric nanoparticles stained with fluorophores differing in their fluorescence lifetimes as a new multiplexing and encoding approach. Proof-of-concept measurements with different sets of lifetime-encoded polystyrene nanoparticles are presented, obtained via staining of preformed particles with visible (vis)- and near-infrared (NIR)-emissive organic dyes, which display very similar absorption and emission spectra to enable excitation and detection at the same wavelengths, yet sufficiently different fluorescence decay kinetics in suspension, thereby minimizing instrumentation costs. Data analysis was performed with a linear combination approach in the lifetime domain. Our results and first cell experiments with these reporter sets underline the suitability of our multiplexing strategy for the discrimination between and the quantification of different labels. This simple and versatile concept can be extended to all types of fluorophores, thereby expanding the accessible time scale, and can be used, e.g., for the design of labels and targeted probes for fluorescence assays and molecular imaging, cellular imaging studies, and barcoding applications, also in conjunction with spectral and intensity encoding. Figure Nanoparticle-based lifetime multiplexing in living cells
Keywords: Fluorescent label; Multiplexing; Optical encoding; Lifetime multiplexing; Fluorescence lifetime imaging FLIM; Nanoparticles
Oligonucleotide microarray chip for the quantification of MS2, ΦX174, and adenoviruses on the multiplex analysis platform MCR 3 by Sandra Lengger; Johannes Otto; Dennis Elsässer; Oliver Schneider; Andreas Tiehm; Jens Fleischer; Reinhard Niessner; Michael Seidel (3323-3334).
Pathogenic viruses are emerging contaminants in water which should be analyzed for water safety to preserve public health. A strategy was developed to quantify RNA and DNA viruses in parallel on chemiluminescence flow-through oligonucleotide microarrays. In order to show the proof of principle, bacteriophage MS2, ΦX174, and the human pathogenic adenovirus type 2 (hAdV2) were analyzed in spiked tap water samples on the analysis platform MCR 3. The chemiluminescence microarray imaging unit was equipped with a Peltier heater for a controlled heating of the flow cell. The efficiency and selectivity of DNA hybridization could be increased resulting in higher signal intensities and lower cross-reactivities of polymerase chain reaction (PCR) products from other viruses. The total analysis time for DNA/RNA extraction, cDNA synthesis for RNA viruses, polymerase chain reaction, single-strand separation, and oligonucleotide microarray analysis was performed in 4–4.5 h. The parallel quantification was possible in a concentration range of 9.6 × 105–1.4 × 1010 genomic units (GU)/mL for bacteriophage MS2, 1.4 × 105–3.7 × 108 GU/mL for bacteriophage ΦX174, and 6.5 × 103–1.2 × 105 for hAdV2, respectively, by using a measuring temperature of 40 °C. Detection limits could be calculated to 6.6 × 105 GU/mL for MS2, 5.3 × 103 GU/mL for ΦX174, and 1.5 × 102 GU/mL for hAdV2, respectively. Real samples of surface water and treated wastewater were tested. Generally, found concentrations of hAdV2, bacteriophage MS2, and ΦX174 were at the detection limit. Nevertheless, bacteriophages could be identified with similar results by means of quantitative PCR and oligonucleotide microarray analysis on the MCR 3.
Keywords: Fluorescence/luminescence; Biochips/high-throughput screening; Bioanalytical methods
Multiplex detection of disease biomarkers using SERS molecular sentinel-on-chip by Hoan T. Ngo; Hsin-Neng Wang; Thomas Burke; Geoffrey S. Ginsburg; Tuan Vo-Dinh (3335-3344).
Developing techniques for multiplex detection of disease biomarkers is important for clinical diagnosis. In this work, we have demonstrated for the first time the feasibility of multiplex detection of genetic disease biomarkers using the surface-enhanced Raman scattering (SERS)-based molecular sentinel-on-chip (MSC) diagnostic technology. The molecular sentinel (MS) sensing mechanism is based upon the decrease of SERS intensity when Raman labels tagged at 3′-ends of MS nanoprobes are physically displaced from the nanowave chip’s surface upon DNA hybridization. The use of bimetallic layer (silver and gold) for the nanowave fabrication was investigated. SERS measurements were performed immediately following a single hybridization reaction between the target single-stranded DNA sequences and the complementary MS nanoprobes immobilized on the nanowave chip without requiring target labeling (i.e., label-free), secondary hybridization, or post-hybridization washing, thus shortening the assay time and reducing cost. Two nucleic acid transcripts, interferon alpha-inducible protein 27 and interferon-induced protein 44-like, are used as model systems for the multiplex detection concept demonstration. These two genes are well known for their critical role in host immune response to viral infection and can be used as molecular signature for viral infection diagnosis. The results indicate the potential of the MSC technology for nucleic acid biomarker multiplex detection. Figure Scheme of two-multiplex detection of complementary target ssDNA sequences using SERS-based molecular sentinel-on-chip diagnostic technology
Keywords: Multiplex DNA detection; Surface-enhanced Raman scattering; Molecular sentinel; Molecular sentinel-on-chip; Nanowave; Metal film over nanosphere
Multivalent chelators for spatially and temporally controlled protein functionalization by Changjiang You; Jacob Piehler (3345-3357).
Site-specific protein modification—e.g. for immobilization or labelling—is a key prerequisite for numerous bioanalytical applications. Although modification by use of short peptide tags is particularly attractive, efficient and bio-orthogonal systems are still lacking. Here, we review the application of multivalent chelators (MCH) for high-affinity yet reversible recognition of oligohistidine (His)-tagged proteins. MCH are based on multiple nitrilotriacetic acid (NTA) moieties grafted on to molecular scaffolds suitable for conjugation to surfaces, probes or other biomolecules. Reversible interaction with the His-tag is mediated via transition metal ions chelated by the NTA moieties. The small size and biochemical compatibility of these recognition units and the possibility of rapid dissociation of the interaction with His-tagged proteins despite sub-nanomolar binding affinity, enable distinct and versatile handling and modification of recombinant proteins. In this review, we briefly introduce the key principles and features of MCH–His-tag interactions and recapitulate the broad spectrum of bioanalytical applications with a focus on quantitative protein interaction analysis on micro or nano-patterned solid surfaces and specific protein labelling in living cells. Figure 1 ᅟ
Keywords: His-tag; Nitrilotriacetic acid; Multivalency; Protein immobilization; Protein–protein interaction analysis; Cell-surface labelling
Characterization of the enhanced peroxidatic activity of amyloid β peptide–hemin complexes towards neurotransmitters by Bettina Neumann; Aysu Yarman; Ulla Wollenberger; Frieder Scheller (3359-3364).
Binding of heme to the amyloid peptides Aβ40/42 is thought to be an initial step in the development of symptoms in the early stages of Alzheimer’s disease by enhancing the intrinsic peroxidatic activity of heme. We found considerably higher acceleration of the reaction for the physiologically relevant neurotransmitters dopamine and serotonin than reported earlier for the artificial substrate 3,3′,5,5′-tetramethylbenzidine (TMB). Thus, the binding of hemin to Aβ peptides might play an even more crucial role in the early stages of Alzheimer’s disease than deduced from these earlier results. To mimic complex formation, a new surface architecture has been developed: The interaction between the truncated amyloid peptide Aβ1-16 and hemin immobilized on an aminohexanethiol spacer on a gold electrode has been analyzed by cyclic voltammetry. The resulting complex has a redox pair with a 25 mV more cathodic formal potential than hemin alone.
Keywords: Alzheimer’s disease; Amyloid beta peptides; Hemin; Neurotransmitters; Peroxidatic activity
A biocatalytic cascade with several output signals—towards biosensors with different levels of confidence by Nataliia Guz; Jan Halámek; James F. Rusling; Evgeny Katz (3365-3370).
The biocatalytic cascade based on enzyme-catalyzed reactions activated by several biomolecular input signals and producing output signal after each reaction step was developed as an example of a logically reversible information processing system. The model system was designed to mimic the operation of concatenated AND logic gates with optically readable output signals generated at each step of the logic operation. Implications include concurrent bioanalyses and data interpretation for medical diagnostics. Figure A biocatalytic cascade with several inputs–outputs was designed for bioanalytical applications providing responses with increasing levels of confidence
Keywords: Bioanalytical methods; Bioassays; Enzymes; Optical sensors; Biocomputing; Logic gates
A novel NMR method for the determination and monitoring of evolution of hydrogen peroxide in aqueous solutions by Constantinos G. Tsiafoulis; Ioannis P. Gerothanassis (3371-3375).
A novel NMR method that allowed the rapid and direct quantitative analysis of hydrogen peroxide in protic solvents was developed. The method was based on the highly deshielded 1H NMR signal of the H2O2 protons (δ ∼ 11.15 ppm at 298 K) in H2O and the combined use of cryoprotective (antifreeze) mixtures of H2O−DMSO-d6, low temperatures (∼260 K), and pH effects in order to achieve minimum proton exchange rate and, thus, sharp 1H line widths. Extremely broad resonances with line widths above 550 Hz at room temperature in H2O were observed in a wide range of pH values, which were reduced below 2 Hz with the use of the above method which resulted in a detection limit of 20.0 μmol L−1 (in tube) even when using very short total experimental time of 10 min. The method was applied in aqueous extract of Greek oregano and in aqueous instant coffee. Line widths below 10 Hz for oregano samples and 17 Hz for instant coffee samples were obtained which resulted (i) in the unequivocal assignment of H2O2 with spiking experiments precluding any confusion with interferences from intrinsic phenolics in the extracts and (ii) in the quantitative investigation of the evolution of H2O2 in real time with parameters easily accessible experimentally. Figure ᅟ
Keywords: Hydrogen peroxide; 1H NMR; Quantitative analysis; Cryoprotective mixtures
Label-free optical biosensor for detection and quantification of the non-steroidal anti-inflammatory drug diclofenac in milk without any sample pretreatment by Sabrina Rau; Urs Hilbig; Günter Gauglitz (3377-3386).
A label-free optical biosensor for detection and quantification of diclofenac in bovine milk has been developed. This was achieved by using reflectometric interference spectroscopy as detection method. In a first step, the immunosensor was developed and optimised in buffer concerning sensitivity, selectivity, stability and reproducibility. By comparing recovery rates—not only the good intra- but also the good inter-chip—reproducibility could be proven. Consequently, the assay was transferred in the more complex matrix milk. By utilising an optimised surface modification and evaluation method, matrix effects could successfully be prevented or circumvented. As a result, the developed immunosensor does not need sample pretreatment at all. By obtaining a limit of detection of 0.112 μg L−1 (0.108 μg kg−1), the capability of the developed biosensor is comparable or better than those of standard detection methods. Moreover, the presented biosensor reaches the range of the maximum residue limit (0.1 μg kg−1) set by the European Union. Thus, for the first time, diclofenac was successfully quantified at relevant levels in milk by using an optical biosensor.
Keywords: Milk analysis; Non-steroidal anti-inflammatory drug; Diclofenac; Reflectometric interference spectroscopy; Label-free biosensor; Matrix effects
Probing the physicochemical interactions of 3-hydroxy-benzo[a]pyrene with different monoclonal and recombinant antibodies by use of fluorescence line-narrowing spectroscopy by Ursula Eisold; Annette Kupstat; Dennis Klier; Philipp-A. Primus; Michael Pschenitza; Reinhard Niessner; Dietmar Knopp; Michael U. Kumke (3387-3394).
Characterization of interactions between antigens and antibodies is of utmost importance both for fundamental understanding of the binding and for development of advanced clinical diagnostics. Here, fluorescence line-narrowing (FLN) spectroscopy was used to study physicochemical interactions between 3-hydroxybenzo[a]pyrene (3OH-BaP, as antigen) and a variety of solvent matrices (as model systems) or anti-polycyclic aromatic hydrocarbon antibodies (anti-PAH). We focused the studies on the specific physicochemical interactions between 3OH-BaP and different, previously obtained, monoclonal and recombinant anti-PAH antibodies. Control experiments performed with non-binding monoclonal antibodies and bovine serum albumin (BSA) indicated that nonspecific interactions did not affect the FLN spectrum of 3OH-BaP. The spectral positions and relative intensities of the bands in the FLN spectra are highly dependent on the molecular environment of the 3OH-BaP. The FLN bands correlate with different vibrational modes of 3OH-BaP which are affected by interactions with the molecular environment (π–π interactions, H-bonding, or van-der-Waals forces). Although the analyte (3OH-BaP) was the same for all the antibodies investigated, different binding interactions could be identified from the FLN spectra on the basis of structural flexibility and conformational multiplicity of the antibodies’ paratopes. Physicochemical interactions of PAH in binding pockets of monoclonal and recombinant antibodies
Keywords: FLNS; Antibody; Paratope; Hapten; Polycyclic aromatic hydrocarbons
Bacterial interactions with proteins and cells relevant to the development of life-threatening endocarditis studied by use of a quartz-crystal microbalance by Stefanie Krajewski; Johannes Rheinlaender; Philip Ries; Denis Canjuga; Carmen Mack; Lutz Scheideler; Tilman E. Schäffer; Jürgen Geis-Gerstorfer; Hans-Peter Wendel; Frank Rupp (3395-3406).
Implant-related infections are a major challenge in clinical routine because of severe complications, for example infective endocarditis (IE). The purpose of this study was to investigate the real-time interaction of S. gordonii with proteins and cells important in the development of IE, in a flow system, by use of a quartz-crystal microbalance (QCM). Acoustic sensors were biologically modified by preconditioning with sterile saliva, platelet-poor plasma (PPP), or platelet-rich plasma (PRP), followed then by perfusion of a bacterial suspension. After perfusion, additional fluorescence and scanning electron microscopic (SEM) studies were performed. The surface structure of S. gordonii was analyzed by atomic force microscopy (AFM). Compared with S. gordonii adhesion on the abiotic sensor surface following normal mass loading indicated by a frequency decrease, adhesion on saliva, PPP, or PRP-conditioned sensors resulted in an increase in frequency. Furthermore, adhesion induced slightly increased damping signals for saliva and PPP-coated sensors but a decrease upon bacterial adhesion to PRP, indicating the formation of a more rigid biofilm. Microscopic analysis confirmed the formation of dense and vital bacterial layers and the aggregation of platelets and bacteria. In conclusion, our study shows that the complex patterns of QCM output data observed are strongly dependent on the biological substrate and adhesion mechanisms of S. gordonii. Overall, QCM sheds new light on the pathways of such severe infections as IE.
Keywords: Quartz-crystal microbalance; Endocarditis; Bacterial adhesion; Saliva; Platelets; Plasma
Combined use of two membrane-potential-sensitive dyes for determination of the Galvani potential difference across a biomimetic oil/water interface by Tatsuya Yoshimura; Hirohisa Nagatani; Toshiyuki Osakai (3407-3414).
The fluorescence behavior of anionic membrane-potential-sensitive dyes, bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)) and bis-(1,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC2(3)), at a biomimetic 1,2-dichloroethane (DCE)/water (W) interface was studied by the mean of potential-modulated fluorescence (PMF) spectroscopy. The respective dyes gave a well-defined PMF signal due to the adsorption/desorption at the DCE/W interface. It was also found that the potentials where the two dyes gave the PMF signals were different by about 100 mV. We then attempted a combined use of the two dyes for determination of the Galvani potential difference across the DCE/W interface. When 40 μM DiBAC4(3) and 15 μM DiSBAC2(3) were initially added to the W phase, distinctly different spectra were obtained for different interfacial potentials. The ratio of the PMF signal intensities at 530 and 575 nm (the fluorescence maximum wavelengths for the respective dyes) showed a clear dependence on the interfacial potential. These results suggested the potential utility of the combined use of two dyes for the determination of membrane potentials in vivo. Figure Combined use of two membrane-potential-sensitive dyes that show different colors of fluorescence emission at their specific adsorption potentials.
Keywords: Membrane-potential-sensitive dye; DiBAC4(3); DiSBAC2(3); Liquid/liquid interface; Potential-modulated fluorescence
Mass spectrometric screening and identification of acidic metabolites in fulvic acid fractions of contaminated groundwater by Carsten Jobelius; Fritz H. Frimmel; Christian Zwiener (3415-3429).
The anaerobic microbial degradation of aromatic and heterocyclic compounds is a prevalent process in contaminated groundwater systems. The introduction of functional groups into the contaminant molecules often results in aromatic and heterocyclic and succinic acids. These metabolites can be used as indicators for prevailing degradation processes. Therefore, there is a strong interest in developing analytical methods for screening and identification of these metabolites. In this study, neutral loss scans (NLS) by liquid chromatography-electrospray ionization/tandem mass spectrometry with losses of CO2 (NL ∆m/z = 44) and C2H4(CO2)2 (NL ∆m/z = 116) were applied for the first time successfully to screen selectively for acidic and succinic metabolites of aromatic and heterocyclic contaminants in two fulvic acid fractions from a contaminated site and a downstream region of a tar oil-polluted groundwater. Identification of these preselected signals was performed by high-resolution mass spectrometry with a liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry instrument. High-resolution mass and mass fragmentation data were then compared with a list of known metabolites from a literature search or matched with chemical databases supported with in silico fragmentation. Based on authentic analytical standards, several compounds from NLS were identified (e.g., 4-hydroxy-3-methylbenzoic acid, benzylsuccinic acid, naphthyl-2-methylsuccinic acid, 2-carboxyindane, and 2-carboxybenzothiophene) and tentatively identified (e.g., benzofuranmethylsuccinic acid and dihydrocarboxybenzothiophene) as aromatic, phenolic, heterocyclic, and succinic acids. The acidic metabolites were found exclusively in the contaminated region of the aquifer which indicates active biodegradation processes and no relevant occurrence of acidic metabolites in the downstream region. Figure ᅟ
Keywords: Mass spectrometric screening; Neutral loss scans; Acidic metabolites; Fulvic acid; Contaminated groundwater
A highly sensitive method for in vitro testing of fluorinated drug candidates using high-resolution continuum source molecular absorption spectrometry (HR-CS MAS) by Irene Würtenberger; Ronald Gust (3431-3442).
We report here the development, optimization, and evaluation of a highly sensitive method for the determination of fluorine in biological matrices employing highresolution continuum source molecular absorption spectrometry (HR-CS MAS), suitable for pharmacological testing of fluorine-containing drug candidates. For this purpose, the most important parameters were studied in detail and subsequently optimized using a multivariate approach based on experimental design methodology. We developed a new approach employing a graphite tube lined with tantalum foil, thereby significantly enhancing sensitivity, while interferences from phosphorus monoxide (PO) molecular absorption due to the complex phosphate-rich matrix were completely eliminated. The limit of detection and the characteristic mass were 5.79 and 6.08 pg F, respectively. In order to evaluate the accuracy of the procedure, a recovery test was performed using spiked samples from three bioassays (i.e., DNA binding, protein binding, and cellular uptake) and the recovery rates ranged from 97.4 to 106.4 %. The proposed method is applicable for preclinical in vitro testing of fluorinated drug molecules and thereby establishes HR-CS atomic absorption spectrometry instrumentation as a universal tool in medicinal chemistry. Figure Determination of fluorine in biological matrices for pharmacological evaluation of fluorinated drug molecules
Keywords: Fluorine; Molecular absorption spectrometry; Graphite furnace high-resolution continuum source AAS; Tantalum lining; Experimental design
Development of an LC-MS/MS method for aromatase inhibitor screening by Myeong Hyeon Park; In Sook Kim; Mi-Sook Dong; Hye Hyun Yoo (3443-3449).
Aromatase (CYP 19A1) is a key steroidogenic enzyme that catalyzes the conversion of androgen to estrogen. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for aromatase inhibitor screening was developed and validated. The substrate androstenedione was incubated with human CYP 19A1 supersomes in the presence of NADPH for 30 min, and estrone formation was determined by LC-MS/MS analysis. Cortisone was used as internal standard. The incubation mixture was extracted using a liquid-liquid extraction method with ethyl acetate. Chromatographic separation was achieved using a C18 column (3.0 × 50 mm, 2.7 μm) with a mobile phase consisting of 0.1 % formic acid/acetonitrile adopting gradient elution at a flow rate of 0.4 mL/min. The mass spectrometer was operated in positive electrospray ionization mode. The precursor-product ion pairs used for multiple reaction monitoring were m/z 287→97 (androstenedione), m/z 271 → 159 (estrone), and m/z 361 → 163 (IS, cortisone). The developed method met the required criteria for the validation of bioanalytical methods. The validated method was successfully applied to evaluate aromatase inhibitory activity of plants extracts of Simaroubaceae. Figure Determination of estrone formation by LC-MS/MS analysis for aromatase inhibitor screening
Keywords: Aromatase; CYP 19A1; Inhibition; LC-MS/MS
Determination of Meserine, a new candidate for Alzheimer’s disease in mice brain by liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic and tissue distribution study by Zhaoxi Zheng; Yabin Tang; Haoyu Lv; Jianrong Xu; Hengyi Zhao; Qiong Xie; Zhuibai Qiu; Hongzhuan Chen; Hao Wang (3451-3458).
A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of Meserine ((−)-meptazinol phenylcarbamate), a novel potent inhibitor of acetylcholinesterase (AChE), was developed, validated, and applied to a pharmacokinetic study in mice brain. The lower limit of quantification (LLOQ) was 1 ng mL−1 and the linear range was 1–1,000 ng mL−1. The analyte was eluted on a Zorbax SB-Aq column (2.1 × 100 mm, 3.5 μm) with the mobile phase composed of methanol and water (70:30, v/v, aqueous phase contained 10 mM ammonium formate and 0.3 % formic acid) using isocratic elution, and monitored by positive electrospray ionization in multiple reaction monitoring (MRM) mode. The flow rate was 0.25 mL min−1. The injection volume was 5 μL and total run time was 4 min. The relative standard deviation (RSD) of intraday and interday variation was 2.49–7.81 and 3.01–7.67 %, respectively. All analytes were stable after 4 h at room temperature and 6 h in autosampler. The extraction recoveries of Meserine in brain homogenate were over 90 %. The main brain pharmacokinetic parameters obtained after intranasal administration were T max = 0.05 h, C max = 462.0 ± 39.7 ng g−1, T 1/2 = 0.4 h, and AUC(0-∞) = 283.1 ± 9.1 ng h g−1. Moreover, Meserine was distributed rapidly and widely into brain, heart, liver, spleen, lung, and kidney tissue. The method is validated and could be applied to the pharmacokinetic and tissue distribution study of Meserine in mice.
Keywords: Meserine; Alzheimer’s disease; LC-MS/MS; Pharmacokinetics; Tissue distribution
Multi-analytical platform metabolomic approach to study miltefosine mechanism of action and resistance in Leishmania by Gisele A. B. Canuto; Emerson A. Castilho-Martins; Marina F. M. Tavares; Luis Rivas; Coral Barbas; Ángeles López-Gonzálvez (3459-3476).
Miltefosine (MT) (hexadecylphosphocholine) was implemented to cope with resistance against antimonials, the classical treatment in Leishmaniasis. Given the scarcity of anti- Leishmania (L) drugs and the increasing appearance of resistance, there is an obvious need for understanding the mechanism of action and development of such resistance. Metabolomics is an increasingly popular tool in the life sciences due to it being a relatively fast and accurate technique that can be applied either with a particular focus or in a global manner to reveal new knowledge about biological systems. Three analytical platforms, gas chromatography (GC), liquid chromatography (LC) and capillary electrophoresis (CE) have been coupled to mass spectrometry (MS) to obtain a broad picture of metabolic changes in the parasite. Impairment of the polyamine metabolism from arginine (Arg) to trypanothione in susceptible parasites treated with MT was in some way expected, considering the reactive oxygen species (ROS) production described for MT. Importantly, in resistant parasites an increase in the levels of amino acids was the most outstanding feature, probably related to the adaptation of the resistant strain for its survival inside the parasitophorous vacuole. Online Abstract Figure ᅟ
Keywords: Miltefosine; Leishmania (L.) donovani ; Metabolic fingerprinting; Mass-spectrometry; Drug action; Resistance
Determination of cis-permethrin, trans-permethrin and associated metabolites in rat blood and organs by gas chromatography–ion trap mass spectrometry by F. Lestremau; M.-E. Willemin; C. Chatellier; S. Desmots; C. Brochot (3477-3487).
An analytical method was developed to measure cis-permethrin and trans-permethrin in different biological rat matrices and fluids (whole blood, red blood cells, plasma, brain, liver, muscle, testes, kidneys, fat and faeces). The method was also suitable for the simultaneous quantification of their associated metabolites [cis-3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (cis-DCCA), trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (trans-DCCA) and 3-phenoxybenzoic acid (3-PBA)] in blood (whole blood, red blood cells, plasma) and liver. The target analytes were derivatised in samples using a methanolic/hydrochloric acid solution and then extracted with toluene. The analysis was performed by gas chromatography, and detection using ion trap tandem mass spectrometry. The selectivity obtained for complex matrices such as rat organs allowed the use of a purification step to be avoided for most of the matrices investigated. In the case of fat, where permethrin is suspected to accumulate, a dedicated purification step was developed. In fluids, the limits of quantification were at the 50 ng/mL level for the parent compounds and 3-PBA and at 25 ng/mL for cis-DCCA and trans-DCCA. For solid matrices excluding fat, the limits of quantification ranged from 50 ng/g for muscle to 100 ng/g for brain and testes for both cis-permethrin and trans-permethrin. The extraction recoveries ranged primarily between 80 and 120 % for the matrix tested. The stability of blood samples was tested through the addition of 1 % v/v formic acid. The methods developed were applied in a toxicokinetic study in adult rats. cis-Permethrin and the metabolites were detected in all corresponding matrices, whereas trans-permethrin was detected only in blood, plasma and faeces.
Keywords: Biological samples; Bioanalytical methods; Gas chromatography; Mass spectrometry/ion trap mass spectrometry; Pesticides/endocrine disruptors
Determination of bis-carboxyethyl germanium sesquioxide by gas chromatography with microwave-induced plasma-atomic emission detection after derivatization with alkyl chloroformates by E. Trikas; G. A. Zachariadis; E. Rosenberg (3489-3496).
Organic germanium compounds, especially Ge-132, more corrctly denoted as bis-beta-carboxyethyl germanium sesquioxide ([Ge(=O)CH2CH2CO2H]2O), are of continued interest as they are said to promote health and display anticancer activity. Although these beneficial effects have never been substantiated by comprehensive clinical studies, this drug can still be obtained through various sources and is usually marketed as a nutritional supplementation rather than an anticancer medication. As the quality standards under which this drug is produced are unknown, the need for an effective quality control of these products arises. To date, Ge-132 is considered generally as a safe compound for application in contrast to inorganic germanium which demonstrates severe renal toxicity. In this paper, a new approach to the determination of Ge-132, based on derivatization by ethyl chloroformate reagent (ECF), in the presence of ethanol and pyridine in the mixture, and subsequent analysis by gas chromatography coupled with microwave-induced plasma-atomic emission detection (GC-MIP-AED), is reported. Reaction conditions of the derivatization procedure were optimized with particular respect to the reagent (ECF) and catalyst (pyridine) concentrations. The proposed method is capable of distinguishing Ge-132 from inorganic germanium. The derivatization procedure was also tested with the use of methyl chloroformate (MCF) as alternative reagent, providing interesting additional information about the nature of the final product and the proposed reaction scheme. Among the two types of chloroformates, i.e., MCF and ECF, the latter proved to be more suitable for the proposed method, providing a calibration curve of superior sensitivity and linearity compared with the one obtained with MCF. The method was applied successfully in three real samples, two food supplements, and one commercially available fertilizer. The analysis of the Ge-132 derivative showed good linearity in the concentration of 1–250 mg L−1 (r 2 = 0.9986) and a satisfactory precision (RSD = 6.8 %), which qualifies the proposed method for the speciation analysis of Ge in various matrices.
Keywords: Gas chromatography; Atomic emission detection; Microwave-induced plasma; Germanium sesquioxide; Ge-132
Validation of a multi-analyte LC–MS/MS method for screening and quantification of 87 psychoactive drugs and their metabolites in hair by Marco Fisichella; Luca Morini; Cristina Sempio; Angelo Groppi (3497-3506).
A multi-analyte method for the detection and quantification of 87 psychoactive drugs (antidepressants, antipsychotics, benzodiazepines, and z-drugs) in human hair has been developed and fully validated using the liquid chromatography–tandem mass spectrometry system. Due to the remarkable increase in requests of hair sample tests (such as for driver’s license renewals, child custody, DFA cases, and postmortem toxicology), we focused on the development of a rapid sample preparation. About 20 mg of hair samples, previously washed and cut into snippets, was ultrasonicated with 700 μl of methanol. Samples were then directly analyzed using a 4000 QTRAP (AB SCIEX, Foster City, CA, USA) with an electrospray ionization (ESI) Turbo VTM Ion Source. The validation criteria parameters were satisfactory and in accordance with the international guidelines. All the compounds tested were successfully detected. One important aspect is the LODs in the low picogram per milligram concentration which may suggest a potential use of this method in cases of detection of single drug exposure. However, the LC–MS/MS method has been successfully applied for the analysis of postmortem cases (n = 9).
Keywords: Psychoactive drugs; Antidepressants; Benzodiazepines; Hair; LC–MS/MS
Copolymer-grafted silica phase from a cation–anion monomer pair for enhanced separation in reversed-phase liquid chromatography by Abul K. Mallik; Hongdeng Qiu; Makoto Takafuji; Hirotaka Ihara (3507-3515).
This work reports a new imidazolium and l-alanine derived copolymer-grafted silica stationary phase for ready separation of complex isomers using high-performance liquid chromatography (HPLC). For this purpose, 1-allyl-3-octadecylimidazolium bromide ([AyImC18]Br) and N-acryloyl-l-alanine sodium salt ([AAL]Na) ionic liquids (IL) monomers were synthesized. Subsequently, the bromide counteranion was exchanged with the 2-(acrylamido)propanoate organic counteranion by reacting the [AyImC18]Br with excess [AAL]Na in water. The obtained IL cation–anion monomer pair was then copolymerized on mercaptopropyl-modified silica (Sil-MPS) via a surface-initiated radical chain-transfer reaction. The selective retention behaviors of polycyclic aromatic hydrocarbons (PAHs), including some positional isomers, steroids, and nucleobases were investigated using the newly obtained Sil-poly(ImC18-AAL), and octadecyl silylated silica (ODS) was used as the reference column. Interesting results were obtained for the separation of PAHs, steroids, and nucleobases with the new organic phase. The results showed that the Sil-poly(ImC18-AAL) presented multiple noncovalent interactions, including hydrophobic, π–π, carbonyl–π, and ion–dipole interactions for the separation of PAHs and dipolar compounds. Only pure water was sufficient as the mobile phase for the separation of the nucleobases. Ten nucleosides and bases were separated, using only water as the mobile phase, within a very short time using the Sil-poly(ImC18-AAL), which is otherwise difficult to achieve using conventional hydrophobic columns such as ODS. The combination of electrostatic and hydrophobic interactions are important for the effective separation of such basic compounds without the use of any organic additive as the eluent on the Sil-poly(ImC18-AAL) column. Figure Separation of pyrimidines including (5) 5-fluorouracil, (6) uracil, (7) 5-bromouracil, (8) thymine, (9) cytosine, and (10) 4,6-diaminopyrimidine on Sil-poly(ImC18-AAL). Mobile phase, pure water; column temperature, 40 °C; flow rate, 1.00 ml min–1
Keywords: Reversed-phase chromatography; Ionic liquid-modified silica; Molecular-shape selectivity; Carbonyl–π interaction; Polycyclic aromatic hydrocarbons; Nucleosides
Development of non-denaturing off-gel isoelectric focusing for the separation of uranium–protein complexes in fish by Guillaume Bucher; Sandrine Frelon; Olivier Simon; Ryszard Lobinski; Sandra Mounicou (3517-3520).
An off-gel non-denaturing isoelectric focusing (IEF) method was developed to separate uranium–biomolecule complexes from biological samples as a first step in a multidimensional metalloproteomic approach. Analysis of a synthetic uranium–bovine serum albumin complex demonstrated the focusing ability of the liquid-phase IEF method and the preservation of most of the uranium–protein interactions. The developed method was applied to gill cytosol prepared from zebrafish (Danio rerio) exposed to depleted uranium. The results were compared in terms of resolution, recovery, and protein identities with those obtained by in-gel IEF using an immobilized pH gradient gel strip.
Keywords: Off-gel isoelectric focusing; Uranium; Protein; Non-denaturing; Zebrafish gill
Mass spectrometric analysis of nanoscale sample volumes extracted from open microchannels after sample preconcentration applied on amyloid beta peptides by Saara Mikkonen; Johan Jacksén; Åsa Emmer (3521-3524).
A new instrumental concept for extraction of nanovolumes from open microchannels (dimensions 150 μm × 50 μm, length 10 mm) manufactured on silicon microchips has been used in combination with a previously developed method for preconcentrating proteins and peptides in the open channels through electromigration. The extracted nanovolumes were further analyzed using nanoelectrospray ionization (nESI) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) directly or with subsequent enzymatic protein digestion in a nanodroplet prior to the MS analysis. Preconcentration of the samples resulted in a 15-fold sensitivity increase in nESI for a neurotensin solution, and using MALDI-MS, amyloid beta (Aβ) peptides could be detected in concentrations down to 1 nM. The method was also successfully applied for detection of cell culture Aβ.
Keywords: Mass spectrometry; Microchannel; Preconcentration; Amyloid beta; MALDI; Nano-ESI