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Analytical and Bioanalytical Chemistry (v.400, #4)

Microorganisms for analysis by Gérald Thouand (pp. 893-894).

received his PhD degree in Microbiology from the University of Nancy, France, in 1993. He is a full Professor in Microbiology at the University of Nantes, head of the CBAC Bacterial Sensors for Control and Analysis laboratory (UMR CNRS 6144 GEPEA), representative for research innovation at the French Ministry of Research (DRRT Pays de La Loire), and auditor with the Institute of Higher Studies for Science and Technology (IHEST, Paris).His research focuses on the environmental monitoring of biodegradation and biotechnology using microbial biosystems. He is primarily involved in the development of biosensors for the detection of chemical pollutants and pathogenic bacteria. A member of the French Society of Microbiology (SFM, France) and the Society for Applied Microbiology (SFAM, UK), he is also associate editor of Frontiers in Microbiology (Frontiers in Microbial Ecotoxicology and Bioremediation).

Whole-cell aquatic biosensors by Evgeni Eltzov; Robert S. Marks (pp. 895-913).

The use of various whole-cell organisms as tools for monitoring water contaminants is reviewed and evaluated. Their suitability and value for assessing a range of environmental problems from the impact of pollution, homeland defense issues, conservation, and the long-term degradation and recovery of ecosystems is discussed. Guidelines are provided for the choice of appropriate bioreporter organisms, transducers, and immobilization methods. Examples of the use of a broad spectrum of whole-cell bioreporters in assessing a variety of environmental problems are summarized.

Keywords: Whole cell; Aquatic biosensors; Toxicity

Are luminescent bacteria suitable for online detection and monitoring of toxic compounds in drinking water and its sources? by Marjolijn Woutersen; Shimshon Belkin; Bram Brouwer; Annemarie P. van Wezel; Minne B. Heringa (pp. 915-929).

Biosensors based on luminescent bacteria may be valuable tools to monitor the chemical quality and safety of surface and drinking water. In this review, an overview is presented of the recombinant strains available that harbour the bacterial luciferase genes luxCDABE, and which may be used in an online biosensor for water quality monitoring. Many bacterial strains have been described for the detection of a broad range of toxicity parameters, including DNA damage, protein damage, membrane damage, oxidative stress, organic pollutants, and heavy metals. Most lux strains have sensitivities with detection limits ranging from milligrams per litre to micrograms per litre, usually with higher sensitivities in compound-specific strains. Although the sensitivity of lux strains can be enhanced by various molecular manipulations, most reported detection thresholds are still too high to detect levels of individual contaminants as they occur nowadays in European drinking waters. However, lux strains sensing specific toxic effects have the advantage of being able to respond to mixtures of contaminants inducing the same effect, and thus could be used as a sensor for the sum effect, including the effect of compounds that are as yet not identified by chemical analysis. An evaluation of the suitability of lux strains for monitoring surface and drinking water is therefore provided.

Keywords: Bioluminescence; Biosensors; Water monitoring; Toxicity; Reporter genes

Development and applications of whole cell biosensors for ecotoxicity testing by Neil F. Pasco; Richard J. Weld; Joanne M. Hay; Ravi Gooneratne (pp. 931-945).

Whole cell biosensors are the focus of considerable and increasing interest worldwide as methods for detecting and quantifying environmental toxicity, including biochemical oxygen demand (BOD), heavy metals, antibiotics, pesticides and herbicides. This review follows the development of whole cell biosensors from attempts to utilise changes in cellular metabolism to determine BOD and general toxicity, through the exploitation of unique metabolic pathways to detect specific toxicants, to the increasingly widespread use of genetic engineering to build new, and modify existing, sensing pathways.

Keywords: Microbial biosensors; Environmental toxicity; Broad response; Specific response; Engineered response

Cell-based electrochemical biosensors for water quality assessment by Florence Lagarde; Nicole Jaffrezic-Renault (pp. 947-964).

During recent decades, extensive industrialisation and farming associated with improper waste management policies have led to the release of a wide range of toxic compounds into aquatic ecosystems, causing a rapid decrease of world freshwater resources and thus requiring urgent implementation of suitable legislation to define water remediation and protection strategies. In Europe, the Water Framework Directive aims to restore good qualitative and quantitative status to all water bodies by 2015. To achieve that, extensive monitoring programmes will be required, calling for rapid, reliable and cost-effective analytical methods for monitoring and toxicological impact assessment of water pollutants. In this context, whole cell biosensors appear as excellent alternatives to or techniques complementary to conventional chemical methods. Cells are easy to cultivate and manipulate, host many enzymes able to catalyse a wide range of biological reactions and can be coupled to various types of transducers. In addition, they are able to provide information about the bioavailability and the toxicity of the pollutants towards eukaryotic or prokaryotic cells. In this article, we present an overview of the use of whole cells, mainly bacteria, yeasts and algae, as sensing elements in electrochemical biosensors with respect to their practical applications in water quality monitoring, with particular emphasis on new trends and future perspectives. In contrast to optical detection, electrochemical transduction is not sensitive to light, can be used for analysis of turbid samples and does not require labelling. In some cases, it is also possible to achieve higher selectivities, even without cell modification, by operating at specific potentials where interferences are limited.

Keywords: Whole cell biosensors; Electrochemical transduction; Water quality; Ecotoxicology; Environmental monitoring

How to design cell-based biosensors using the sol–gel process by Christophe Depagne; Cécile Roux; Thibaud Coradin (pp. 965-976).

Inorganic gels formed using the sol–gel process are promising hosts for the encapsulation of living organisms and the design of cell-based biosensors. However, the possibility to use the biological activity of entrapped cells as a biological signal requires a good understanding and careful control of the chemical and physical conditions in which the organisms are placed before, during, and after gel formation, and their impact on cell viability. Moreover, it is important to examine the possible transduction methods that are compatible with sol–gel encapsulated cells. Through an updated presentation of the current knowledge in this field and based on selected examples, this review shows how it has been possible to convert a chemical technology initially developed for the glass industry into a biotechnological tool, with current limitations and promising specificities. Figure Optical fluorescence image of living bacteria in silica thin films

Keywords: Encapsulation; Living cells; Sol–gel; Biosensors; Silica

Bacterial spores as platforms for bioanalytical and biomedical applications by Leslie D. Knecht; Patrizia Pasini; Sylvia Daunert (pp. 977-989).

Genetically engineered bacteria-based sensing systems have been employed in a variety of analyses because of their selectivity, sensitivity, and ease of use. These systems, however, have found limited applications in the field because of the inability of bacteria to survive long term, especially under extreme environmental conditions. In nature, certain bacteria, such as those from Clostridium and Bacillus genera, when exposed to threatening environmental conditions are capable of cocooning themselves into a vegetative state known as spores. To overcome the aforementioned limitation of bacterial sensing systems, the use of microorganisms capable of sporulation has recently been proposed. The ability of spores to endow bacteria-based sensing systems with long lives, along with their ability to cycle between the vegetative spore state and the germinated living cell, contributes to their attractiveness as vehicles for cell-based biosensors. An additional application where spores have shown promise is in surface display systems. In that regard, spores expressing certain enzymes, proteins, or peptides on their surface have been presented as a stable, simple, and safe new tool for the biospecific recognition of target analytes, the biocatalytic production of chemicals, and the delivery of biomolecules of pharmaceutical relevance. This review focuses on the application of spores as a packaging method for whole-cell biosensors, surface display of recombinant proteins on spores for bioanalytical and biotechnological applications, and the use of spores as vehicles for vaccines and therapeutic agents. Figure Bacterial spores have been utilized in biotechnological applications due to their innate stability under normal and extreme environmental conditions. Specifically, the spores have been employed to preserve whole-cell sensing systems (top panel) and to surface display heterologous proteins (bottom panel) in bioanalytical and biomedical applications.

Keywords: Spore; Whole-cell biosensors; Spore surface display

Bacteriophage reporter technology for sensing and detecting microbial targets by Abby E. Smartt; Steven Ripp (pp. 991-1007).

Bacteriophages (phages) are bacterial viruses evolutionarily tuned to very specifically recognize, infect, and propagate within only a unique pool of host cells. Knowledge of these phage host ranges permits one to devise diagnostic tests based on phage–host recognition profiles. For decades, fundamental phage typing assays have been used to identify bacterial pathogens on the basis of the ability of phages to kill, or lyse, the unique species, strain, or serovar to which they are naturally targeted. Over time, and with a better understanding of phage–host kinetics and the realization that there exists a phage specific for nearly any bacterial pathogen of clinical, foodborne, or waterborne consequence, a variety of improved, rapid, sensitive, and easy-to-use phage-mediated detection assays have been developed. These assays exploit every stage of the phage recognition and infection cycle to yield a wide variety of pathogen monitoring, detection, and enumeration formats that are steadily advancing toward new biosensor integrations and advanced sensing technologies.

Keywords: Bacteriophage; Bioreporter; Biosensor; Pathogen; Phage; Reporter gene

Microscreening toxicity system based on living magnetic yeast and gradient chips by Javier García-Alonso; Rawil F. Fakhrullin; Vesselin N. Paunov; Zheng Shen; Joerg D. Hardege; Nicole Pamme; Stephen J. Haswell; Gillian M. Greenway (pp. 1009-1013).

There is an increasing demand for easy and cost-effective methods to screen the toxicological impact of the growing number of chemical mixtures being generated by industry. Such a screening method has been developed using viable, genetically modified green fluorescent protein (GFP) reporter yeast that was magnetically functionalised and held within a microfluidic device. The GFP reporter yeast was used to detect genotoxicity by monitoring the exposure of the cells to a well-known genotoxic chemical (methyl methane sulfonate, MMS). The cells were magnetised using biocompatible positively charged PAH-stabilised magnetic nanoparticles with diameters around 15 nm. Gradient mixing was utilised to simultaneously expose yeast to a range of concentrations of toxins, and the effective fluorescence emitted from the produced GFP was measured. The magnetically enhanced retention of the yeast cells, with their facile subsequent removal and reloading, allowed for very convenient and rapid toxicity screening of a wide range of chemicals. This is the first report showing magnetic yeast within microfluidic devices in a simple bioassay, with potential applications to other types of fluorescent reporter yeast in toxicological and biomedical research. The microfluidic chip offers a simple and low-cost screening test that can be automated to allow multiple uses (adapted to different cell types) of the device on a wide range of chemicals and concentrations. Multi-concentration chemical toxicity screening within a microsystem using a fluorescent reporter yeast, with the fluorescence increasing as the concentration of the genotoxic compound increases.

Keywords: Toxicity screening; GFP reporter yeast; Magnetic retention; Microfluidic devices

Free Ca²⁺ as an early intracellular biomarker of exposure of cyanobacteria to environmental pollution by Ana Lilia Barrán-Berdón; Ismael Rodea-Palomares; Francisco Leganés; Francisca Fernández-Piñas (pp. 1015-1029).

Calcium functions as a versatile messenger in a wide variety of eukaryotic and prokaryotic cells. Cyanobacteria are photoautotrophs which have a great ecological impact as primary producers. Our research group has presented solid evidence of a role of calcium in the perception of environmental changes by cyanobacteria and their acclimation to these changes. We constructed a recombinant strain of the freshwater cyanobacterium Anabaena sp. PCC 7120 that constitutively expresses the calcium-binding photoprotein apoaequorin, enabling in-vivo monitoring of any fluctuation in the intracellular free calcium concentration of the cyanobacterium in response to any environmental stimulus. The “Ca²⁺ signature” is the combination of changes in all Ca²⁺ signal properties (magnitude, duration, frequency, source of the signal) produced by a specific stimulus. We recorded and analyzed the Ca²⁺ signatures generated by exposure of the cyanobacterium to different groups of environmental pollutants, for example cations, anions, organic solvents, naphthalene, and pharmaceuticals. We found that, in general, each group of tested chemicals triggered a specific calcium signature in a reproducible and dose-dependent manner. We hypothesize that these Ca²⁺ signals may be related to the cellular mechanisms of pollutant perception and ultimately to their toxic mode of action. We recorded Ca²⁺ signals triggered by binary mixtures of pollutants and a signal induced by a real wastewater sample which could be mimicked by mixing its main constituents. Because Ca²⁺ signatures were induced before toxicity was evident, we propose that intracellular free Ca²⁺ may serve as an early biomarker of exposure to environmental pollution. Figure

Keywords: Aequorin; Biomarker; Ca²⁺ signature; Cyanobacterium; Environmental pollution; Pollutant interaction

A pH-based biosensor for detection of arsenic in drinking water by K. de Mora; N. Joshi; B. L. Balint; F. B. Ward; A. Elfick; C. E. French (pp. 1031-1039).

Arsenic contaminated groundwater is estimated to affect over 100 million people worldwide, with Bangladesh and West Bengal being among the worst affected regions. A simple, cheap, accurate and disposable device is required for arsenic field testing. We have previously described a novel biosensor for arsenic in which the output is a change in pH, which can be detected visually as a colour change by the use of a pH indicator. Here, we present an improved formulation allowing sensitive and accurate detection of less than 10 ppb arsenate with static overnight incubation. Furthermore, we describe a cheap and simple high-throughput system for simultaneous monitoring of pH in multiple assays over time. Up to 50 samples can be monitored continuously over the desired time period. Cells can be stored and distributed in either air-dried or freeze-dried form. This system was successfully tested on arsenic-contaminated groundwater samples from the South East region of Hungary. We hope to continue to develop this sensor to produce a device suitable for field trials. Figure Arsenic assays using bromothymol blue as a pH indicator. Each arsenic concentration was tested in triplicate on two separated days. Samples (50 μl inoculum) were statically incubated at 37 °C for a period of 65 h. Blue represents a safe level of arsenic while yellow indicates contamination. This figure shows the biosensor tubes in increasing arsenic concentrations at t = 0, 24, 36, 42, 48 and 60 h where the error represents the standard error of six replicates

Keywords: Biosensors; Groundwater; Arsenic; Assay; BioBricks

Luminescent bacteria-based sensing method for methylmercury specific determination by Anne Rantala; Mikko Utriainen; Nitesh Kaushik; Marko Virta; Anna-Liisa Välimaa; Matti Karp (pp. 1041-1049).

A bacterial biosensor method for the selective determination of a bioavailable organomercurial compound, methylmercury, is presented. A recombinant luminescent whole-cell bacterial strain responding to total mercury content in samples was used. The bacterial cells were freeze-dried and used as robust, reagent-like compounds, without batch-to-batch variations. In this bacteria-based sensing method, luciferase is used as a reporter, which requires no substrate additions, therefore allowing homogenous, real-time monitoring of the reporter gene expression. A noninducible, constitutively light-producing control bacterial strain was included in parallel for determining the overall cytotoxicity of the samples. The specificity of the total mercury sensor Escherichia coli MC1061 (pmerRBlux) bacterial resistance system toward methylmercury is due to a coexpressed specific enzyme, organomercurial lyase. This enzyme mediates the cleavage of the carbon–mercury bond of methylmercury to yield mercury ions, which induce the reporter genes and produce a self-luminescent cell. The selective analysis of methylmercury with the total mercury strain is achieved by specifically chelating the inorganic mercury species from the sample using an optimized concentration of EDTA as a chelating agent. After the treatment with the chelating agent, a cross-reactivity of 0.2% with ionic mercury was observed at nonphysiological ionic mercury concentrations (100 nM). The assay was optimized to be performed in 3 h but results can already be read after 1 h incubation. Total mercury strain E. coli MC1061 (pmerRBlux) has been shown to be highly sensitive and capable of determining methylmercury at a subnanomolar level in optimized assay conditions with a very high dynamic range of two decades. The limit of detection of 75 ng/l (300 pM) allows measurement of methylmercury even from natural samples.

Keywords: Mercury; Luminescent bacterial sensors; Bioluminescence; Bioavailability; Chelating agent; EDTA; Environment

A multi-channel bioluminescent bacterial biosensor for the on-line detection of metals and toxicity. Part I: design and optimization of bioluminescent bacterial strains by Thomas Charrier; Marie-José Durand; Sulivan Jouanneau; Michel Dion; Mimma Pernetti; Denis Poncelet; Gérald Thouand (pp. 1051-1060).

This study describes the construction of inducible bioluminescent strains via genetic engineering along with their characterization and optimization in the detection of heavy metals. Firstly, a preliminary comparative study enabled us to select a suitable carbon substrate from pyruvate, glucose, citrate, diluted Luria–Bertani, and acetate. The latter carbon source provided the best induction ratios for comparison. Results showed that the three constructed inducible strains, Escherichia coli DH1 pBzntlux, pBarslux, and pBcoplux, were usable when conducting a bioassay after a 14-h overnight culture at 30 °C. Utilizing these sensors gave a range of 12 detected heavy metals including several cross-detections. Detection limits for each metal were often close to and sometimes lower than the European standards for water pollution. Finally, in order to maintain sensitive bacteria within the future biosensor-measuring cell, the agarose immobilization matrix was compared to polyvinyl alcohol (PVA). Agarose was selected because the detection limits of the bioluminescent strains were not affected, in contrast to PVA. Specific detection and cross-detection ranges determined in this study will form the basis of a multiple metals detection system by the new multi-channel Lumisens3 biosensor.

Keywords: Bioluminescence; Heavy metals; Bacteria; Detection; Immobilization

A multi-channel bioluminescent bacterial biosensor for the on-line detection of metals and toxicity. Part II: technical development and proof of concept of the biosensor by Thomas Charrier; Cyrille Chapeau; Loubna Bendria; Pascal Picart; Philippe Daniel; Gérald Thouand (pp. 1061-1070).

This research study deals with the on-line detection of heavy metals and toxicity within the context of environmental pollution monitoring. It describes the construction and the proof of concept of a multi-channel bioluminescent bacterial biosensor in immobilized phase: Lumisens3. This new versatile device, designed for the non-stop analysis of water pollution, enables the insertion of any bioluminescent strains (inducible or constitutive), immobilized in a multi-well removable card. The technical design of Lumisens3 has benefited from both a classical and a robust approach and includes four main parts: (1) a dedicated removable card contains 64 wells, 3 mm in depth, arranged in eight grooves within which bacteria are immobilized, (2) this card is incubated on a Pelletier block with a CCD cooled camera on top for bioluminescence monitoring, (3) a fluidic network feeds the card with the sample to be analyzed and finally (4) a dedicated computer interface, BIOLUX 1.0, controls all the elements of the biosensor, allowing it to operate autonomously. The proof of concept of this biosensor was performed using a set of four bioluminescent bacteria (Escherichia coli DH1 pBzntlux, pBarslux, pBcoplux, and E. coli XL1 pBfiluxCDABE) in the on-line detection of CdCl₂ 0.5 μM and As₂O₃ 5 μM from an influent. When considering metals individually, the “fingerprints” from the biosensor were as expected. However, when metals were mixed together, cross reaction and synergistic effects were detected. This biosensor allowed us to demonstrate the simultaneous on-line cross detection of one or several heavy metals as well as the measurement of the overall toxicity of the sample.

Keywords: Multi-channel biosensor; Bacteria; Heavy metals; Bioluminescence; Toxicity

Upgrading bioluminescent bacterial bioreporter performance by splitting the lux operon by Sharon Yagur-Kroll; Shimshon Belkin (pp. 1071-1082).

Bioluminescent bacterial bioreporters harbor a fusion of bacterial bioluminescence genes (luxCDABE), acting as the reporting element, to a stress-response promoter, serving as the sensing element. Upon exposure to conditions that activate the promoter, such as an environmental stress or the presence of an inducing chemical, the promoter::reporter fusion generates a dose-dependent bioluminescent signal. In order to improve bioluminescent bioreporter performance we have split the luxCDABE genes of Photorhabdus luminescens into two smaller functional units: luxAB, that encode for the luciferase enzyme, which catalyzes the luminescence reaction, and luxCDE that encode for the enzymatic complex responsible for synthesis of the reaction’s substrate, a long-chain aldehyde. The expression of each subunit was put under the control of either an inducible stress-responsive promoter or a synthetic constitutive promoter, and different combinations of the two units were tested for their response to selected chemicals in Escherichia coli. In all cases tested, the split combinations proved to be superior to the native luxCDABE configuration, suggesting an improved efficiency in the transcription and/or translation of two small gene units instead of a larger one with the same genes. The best combination was that of an inducible luxAB and a constitutive luxCDE, indicating that aldehyde availability is limited when the five genes are expressed together in E. coli, and demonstrating that improved biosensor performance may be achieved by rearrangement of the lux operon genes. Figure Splitting the Photorhabdus luminescens luxCDABE genes into two independently controlled units in E. coli dramatically enhaced microbial bioreporter performance

Keywords: Biosensors; Bioluminesence; luxCDABE

A new bacterial biosensor for trichloroethylene detection based on a three-dimensional carbon nanotubes bioarchitecture by Mouna Hnaien; Florence Lagarde; Joan Bausells; Abdelhamid Errachid; Nicole Jaffrezic-Renault (pp. 1083-1092).

Trichloroethylene (TCE), a suspected human carcinogen, is one of the most common volatile groundwater contaminants. Many different methodologies have already been developed for the determination of TCE and its degradation products in water, but most of them are costly, time-consuming and require well-trained operators. In this work, a fast, sensitive and miniaturised whole cell conductometric biosensor was developed for the determination of trichloroethylene. The biosensor assembly was prepared by immobilising Pseudomonas putida F1 bacteria (PpF1) at the surface of gold interdigitated microelectrodes through a three-dimensional alkanethiol self-assembly monolayer/carbon nanotube architecture functionalised with Pseudomonas antibodies. The biosensor response was linear from 0.07 to 100 μM of TCE (9–13,100 μg L⁻¹). No significant loss of the enzymatic activity was observed after 5 weeks of storage at 4 °C in the M457 pH 7 defined medium (two or three measurements per week). Ninety-two per cent of the initial signal still remained after 7 weeks. The biosensor response to TCE was not significantly affected by cis-1,2-dichloroethylene and vinyl chloride and, in a limited way, by phenol. Toluene was the major interference found. The bacterial biosensor was successfully applied to the determination of TCE in spiked groundwater samples and in six water samples collected in an urban industrial site contaminated with TCE. Gas chromatography–mass spectrometric analysis of these samples confirmed the biosensor measurements. Figure General view of the interdigitated microelectrodes

Keywords: Trichloroethylene; Whole cell biosensor; Pseudomonas putida F1; Interdigitated microelectrodes; Single-wall carbon nanotubes; Self-assembly monolayer

In situ detection of aromatic compounds with biosensor Pseudomonas putida cells preserved and delivered to soil in water-soluble gelatin capsules by Aitor de las Heras; Víctor de Lorenzo (pp. 1093-1104).

While many types of bacteria have been engineered to produce an optical output in response to given analytes in a culture, their use for extensive, in situ monitoring of distinct chemical species in soil is hampered by a dearth of practicable spreading schemes. In this work, we report and validate a comprehensive system for the long-term preservation of Pseudomonas putida cells genetically designed for biosensing benzene, toluene, ethylbenzene, and xylenes (BTEX) in soil, along with a procedure to formulate, spread, and vigorously activate such bacteria at the desired site and occasion. To this end, various known lyoprotectants were tested for promoting the long-term maintenance of biosensor cells with quite variable outcomes. While a formulation of inositol and maltodextrines was optimal for preservation of freeze-dried BTEX-sensing bacteria, adsorption of P. putida cells to corncob powder (an abundant residue of the corn industry) endowed the resulting material with a lasting viability at ambient conditions. In any case, the thereby preserved bacterial biomass acquired physical and mechanical properties adequate for formulating the biosensor agent in water-soluble but otherwise hard dry gelatine capsules with a long shelf life. When such capsules were spread in a soil microcosm and subsequently liquefied with water or high humidity, the released microorganisms formed spots that gave an intense luminiscent signal upon exposure to effectors of the sensor circuit implanted in the chromosome of the P. putida strain. We argue that the procedures described here can facilitate implementation of wide-area biological detection strategies for revealing the location of toxic or perilous chemicals.

Keywords: Pseudomonas putida ; XylR; Biosensors; Desiccation; Encapsulation

Source inference of exogenous gamma-hydroxybutyric acid (GHB) administered to humans by means of carbon isotopic ratio analysis: novel perspectives regarding forensic investigation and intelligence issues by François Marclay; Christophe Saudan; Julie Vienne; Mehdi Tafti; Martial Saugy (pp. 1105-1112).

γ-Hydroxybutyric acid (GHB) is an endogenous short-chain fatty acid popular as a recreational drug due to sedative and euphoric effects, but also often implicated in drug-facilitated sexual assaults owing to disinhibition and amnesic properties. Whilst discrimination between endogenous and exogenous GHB as required in intoxication cases may be achieved by the determination of the carbon isotope content, such information has not yet been exploited to answer source inference questions of forensic investigation and intelligence interests. However, potential isotopic fractionation effects occurring through the whole metabolism of GHB may be a major concern in this regard. Thus, urine specimens from six healthy male volunteers who ingested prescription GHB sodium salt, marketed as Xyrem®, were analysed by means of gas chromatography/combustion/isotope ratio mass spectrometry to assess this particular topic. A very narrow range of δ¹³C values, spreading from −24.81‰ to −25.06‰, was observed, whilst mean δ¹³C value of Xyrem® corresponded to −24.99‰. Since urine samples and prescription drug could not be distinguished by means of statistical analysis, carbon isotopic effects and subsequent influence on δ¹³C values through GHB metabolism as a whole could be ruled out. Thus, a link between GHB as a raw matrix and found in a biological fluid may be established, bringing relevant information regarding source inference evaluation. Therefore, this study supports a diversified scope of exploitation for stable isotopes characterized in biological matrices from investigations on intoxication cases to drug intelligence programmes.

Keywords: Gamma-hydroxybutyric acid; Gamma-butyrolactone; Sodium oxybate; Isotope ratio mass spectrometry; Urine; Source inference; Forensic

Multi-walled carbon nanotubes–dispersive solid-phase extraction combined with nano-liquid chromatography for the analysis of pesticides in water samples by María Asensio-Ramos; Giovanni D’Orazio; Javier Hernandez-Borges; Anna Rocco; Salvatore Fanali (pp. 1113-1123).

In this work, the simultaneous separation of a group of 12 pesticides (carbaryl, fensulfothion, mecoprop, fenamiphos, haloxyfop, diclofop, fipronil, profenofos, fonofos, disulfoton, nitrofen, and terbufos) by nano-liquid chromatography with UV detection is described. For the analyses, a 100 μm internal diameter capillary column packed with silica modified with phenyl groups was used. Experimental parameters, including the use of a trapping column for increasing the sensitivity, were optimized and validated. A preliminary study of the applicability of a rapid and practical dispersive solid-phase extraction (DSPE) procedure was developed for the extraction of some of these pesticides (carbaryl, fensulfothion, fenamiphos, fipronil, profenofos, fonofos, disulfoton, nitrofen, and terbufos) from Milli-Q water samples using multi-walled carbon nanotubes (MWCNTs). The method was validated through a recovery study at three different levels of concentration, obtaining limits of detection in the range 0.016–0.067 μg/L (below European Union maximum residue limits) for the majority of the pesticides. In this work, MWCNTs were reused up to five times, representing an important reduction of the waste of stationary phase. Furthermore, DSPE permitted a clear diminution of the total sample treatment time with respect to conventional SPE.

Keywords: Nano-liquid chromatography; Pesticides; Multi-walled carbon nanotubes; Dispersive solid-phase extraction

Flow injection measurements of S-nitrosothiols species in biological samples using amperometric nitric oxide sensor and soluble organoselenium catalyst reagent by Chuncui Huang; Elizabeth Brisbois; Mark E. Meyerhoff (pp. 1125-1135).

A novel flow injection analysis (FIA) system suitable for measurement of S-nitrosothiols (RSNOs) in blood plasma is described. In the proposed (FIA) system, samples and standards containing RSNO species are injected into a buffer carrier stream that is mixed with the reagent stream containing 3,3′-dipropionicdiselenide (SeDPA) and glutathione (GSH). SeDPA has been shown previously to catalytically decompose RSNOs in the presence of a reducing agent, such as GSH, to produce nitric oxide (NO). The liberated NO is then detected downstream by an amperometric NO sensor. This sensor is prepared using an electropolymerized m-phenylenediamine (m-PD)/resorcinol and Nafion composite films at the surface of a platinum electrode. Using optimized flow rates and reagent concentrations, detection of various RSNOs at levels in the range of 0.25–20 μM is possible. For plasma samples, detection of background sensor interference levels within the samples must first be carried out using an identical FIA arrangement, but without the added SeDPA and GSH reagents. Subtraction of this background sensor current response allows good analytical recovery of RSNOs spiked into animal plasma samples, with recoveries in the range of 90.4–101.0%. Figure Samples and standards containing S-nitrosothiols (RSNOs) are injected into the proposed flow injection analysis system and react with 3,3′-dipropionicdiselenide (SeDPA) and glutathione (GSH) to liberate nitric oxide (NO), which is detected downstream via an amperometric NO sensor

Keywords: Flow injection analysis; Plasma; S-nitrosothiols (RSNOs); Nitric oxide sensors

Capillary electrophoresis-mass spectrometry analysis of trehalose-6-phosphate in Arabidopsis thaliana seedlings by T. L. Delatte; H. Schluepmann; S. C. M. Smeekens; G. J. de Jong; G. W. Somsen (pp. 1137-1144).

Trehalose-6-phosphate (T6P) is an intermediate in the plant metabolic pathway that results in trehalose production. T6P has been shown to inhibit the sucrose nonfermenting-1-related protein kinase 1, which is a major regulator of metabolism. The quantitation of T6P has proven difficult due to the complexity of the plant matrix and the low abundance of T6P in plant tissues. The aim of this work was to develop a quantitation method for T6P present in Arabidopsis tissues, with capillary electrophoresis (CE) coupled to electrospray ionization-mass spectrometry (MS) with a sheath liquid (SL) interface. The CE-MS method was first optimized with respect to T6P signal intensity and separation of isomers by studying the composition of the background electrolyte (BGE) and SL. The use of triethylamine (TEA) in the BGE was favorable, providing separation of T6P from sucrose-6-phosphate and minimizing ionization suppression. Replacing ammonium acetate with TEA enhanced T6P signal intensities more than four times. The optimized method allowed quantification of T6P in plant extracts with good linearity (r ² > 0.99) within a biologically relevant concentration range. The limit of quantification was 80 nM in Arabidopsis extracts, corresponding to 33 pmol/g plant fresh weight. The CE-MS method was applied to the determination of T6P in seedlings from wild type (WT) Arabidopsis and mutants lacking the trehalase AtTRE1, tre1-1, challenged with trehalose or sorbitol. T6P accumulation in tre1-1 plants grown on sorbitol was about twice the level of T6P found in WT. CE-MS is shown to be a fast and reliable technique to analyze phosphodisaccharides for seedling extracts. The low sample volume requirement of CE and its direct MS coupling makes it an attractive alternative for anion-exchange liquid chromatography–MS.

Keywords: Trehalose-6-phosphate; Capillary electrophoresis; Mass spectrometry; Arabidopsis thaliana ; Trehalase; Phosphodisaccharides

Carbon nano-strings as reporters in lateral flow devices for DNA sensing by hybridization by Despina P. Kalogianni; Lemonia M. Boutsika; Panagiota G. Kouremenou; Theodore K. Christopoulos; Penelope C. Ioannou (pp. 1145-1152).

Presently, there is a growing interest in the development of lateral flow devices for nucleic acid analysis that enable visual detection of the target sequence (analyte) while eliminating several steps required for pipetting, incubation, and washing out the excess of reactants. In this paper, we present, for the first time, lateral flow tests exploiting oligonucleotide-functionalized and antibody-functionalized carbon nanoparticles (carbon nano-strings, CBNS) as reporters that enable confirmation of the target DNA sequence by hybridization. The CBNS reporters were applied to (a) the detection of PCR products and (b) visual genotyping of single nucleotide polymorphisms in human genomic DNA. Biotinylated PCR product was hybridized with a dA-tailed probe. In one assay configuration, the hybrid is captured at the test zone of the strip by immobilized streptavidin and detected by (dT) 30 -CBNS. In a second configuration, the hybrids are captured from immobilized (dA) strands and detected by antibiotin-CBNS. As low as 2.5 fmol of amplified DNA can be detected. For visual genotyping, allele-specific primers with a 5′ oligo(dA) segment are extended by DNA polymerase with a concomitant incorporation of biotin moieties. Extension products are detected either by (dT) 30 -CBNS or by antibiotin-CBNS. Only three cycles of extension reaction are sufficient for detection. No purification of the PCR products or the extension product is required. Figure

Keywords: Lateral flow; DNA; Hybridization; Carbon nanoparticles; Single nucleotide polymorphisms

Using multidimensional projection techniques for reaching a high distinguishing ability in biosensing by Fernando V. Paulovich; Rafael M. Maki; Maria C. F. de Oliveira; Marcelle C. Colhone; Fabiana R. Santos; Vanessa Migliaccio; Pietro Ciancaglini; Katia R. Perez; Rodrigo G. Stabeli; Ângelo C. Perinoto; Osvaldo N. Oliveira Jr; Valtencir Zucolotto (pp. 1153-1159).

Recent advances in the control of molecular engineering architectures have allowed unprecedented ability of molecular recognition in biosensing, with a promising impact for clinical diagnosis and environment control. The availability of large amounts of data from electrical, optical, or electrochemical measurements requires, however, sophisticated data treatment in order to optimize sensing performance. In this study, we show how an information visualization system based on projections, referred to as Projection Explorer (PEx), can be used to achieve high performance for biosensors made with nanostructured films containing immobilized antigens. As a proof of concept, various visualizations were obtained with impedance spectroscopy data from an array of sensors whose electrical response could be specific toward a given antibody (analyte) owing to molecular recognition processes. In addition to discussing the distinct methods for projection and normalization of the data, we demonstrate that an excellent distinction can be made between real samples tested positive for Chagas disease and Leishmaniasis, which could not be achieved with conventional statistical methods. Such high performance probably arose from the possibility of treating the data in the whole frequency range. Through a systematic analysis, it was inferred that Sammon’s mapping with standardization to normalize the data gives the best results, where distinction could be made of blood serum samples containing 10⁻⁷ mg/mL of the antibody. The method inherent in PEx and the procedures for analyzing the impedance data are entirely generic and can be extended to optimize any type of sensor or biosensor.

Keywords: Biosensors; Nanostructured films; Leishmaniasis; Chagas disease; Projection techniques; Information visualization

Mid-IR fiber-optic reflectance spectroscopy for identifying the finish on wooden furniture by T. Poli; O. Chiantore; M. Nervo; A. Piccirillo (pp. 1161-1171).

Mid-IR fiber-optic reflectance spectroscopy (FORS) is a totally noninvasive infrared analytical technique allowing the investigation of artworks without the need for any sampling. The development and optimization of this analytical methodology can provide a tool that is capable of supporting conservators during the first steps of their interventions, yielding fast results and dramatically reducing the number of samples needed to identify the materials involved. Furthermore, since reflection IR spectra suffer from important spectral anomalies that complicate accurate spectral interpretation, it is important to characterize known reference materials and substrates in advance. This work aims to verify the possibility of investigating and identifying the most widely used wood finishes by means of fiber-optic (chalcogenide and metal halides) mid-infrared spectroscopy. Two historically widely employed wood finishes (beeswax, shellac) and two modern ones (a hydrogenated hydrocarbon resin and a microcrystalline wax) were investigated in an extended IR range (from 1000 to 6000 cm⁻¹) with reflectance spectroscopy and with FORS. The broad spectral response of the MCT detector was exploited in order to include overtones and combination bands from the NIR spectral range in the investigation. The reflectance spectra were compared with those collected in transmission mode in order to highlight modifications to shapes and intensities, to assign absorptions, and finally to select “marker” bands indicating the presence of certain finishing materials, even when applied onto a substrate such as wood, which shows many absorptions in the mid-infrared region. After the characterization, the different products were applied to samples of aged pear wood and investigated with the same techniques in order to check the ability of mid-IR FORS to reveal the presence and composition of the product on the wooden substrate.

Keywords: FTIR; Fiber-optic reflectance spectroscopy; Wood treatment products; Regalrez; Wood finishing

Raman and FTIR microspectroscopic study of the alteration of Chinese tung oil and related drying oils during ageing by Anna Schönemann; Howell G. M. Edwards (pp. 1173-1180).

Tung oil is favoured in applications such as historic wood consolidation or as varnish component that require a rapid-drying medium compared with linseed oil and other analogues such as walnut oil and poppy seed oil. The Raman spectra of tung oil and artificially aged specimens have been obtained and indicate that severe degradation of the C=C unsaturation sites occurs compared with the slower-drying linseed oil. Characteristic spectral signatures of fresh tung oil have been identified which provide diagnostic discrimination between this oil and others used in the preparation and preservation of artworks. Mid-infrared spectra of aged tung oils have served to identify the formation of acidic functionalities which could affect associated pigments and substrates in artwork. Comparative spectra are also reported for a range of other oils such as walnut seed, poppy and sunflower seed oils.

Keywords: Raman spectroscopy; Infrared spectroscopy; Aged oils; Drying oils; Tung oil; Linseed oil

Simultaneous quantification of loureirin A and loureirin B in rat urine, feces, and bile by HPLC-MS/MS method and its application to excretion study by Yushi Zhang; Yongzhi Li; Bo Chen; Yulin Deng; Yujuan Li (pp. 1181-1187).

A simple HPLC-MS/MS method for simultaneous determination of loureirin A and loureirin B in rat urine, feces, and bile after oral administration of 10.6 g/kg of longxuejie (one rare traditional Chinese medicinal herb) was developed for the first time. The analytes and buspirone (internal standard) were separated on a C₅ column with acetonitrile–water (containing 0.1% formic acid) as mobile phase at a flow rate of 0.4 min/mL. The detector was a Q-trap™ mass spectrometer with an electrospray ionization interface operating in the multiple reaction monitoring mode. Calibration curves of loureirin A in rat urine, feces, and bile were linear over the concentration range of 1.00–5,000 ng/mL. Loureirin B in rat urine, feces, and bile ranged between 0.08 and 20, 0.20 and 20, and 0.10 and 500 ng/mL, respectively. Validation revealed that the method was specific, accurate, and precise. The fully validated method was applied to the excretion study of loureirin A and loureirin B in rats. After oral administration of 10.6 g/kg longxuejie, cumulative excretion amount of loureirin A and loureirin B in rat urine were 2.94 ± 0.81 and 0.36 ± 0.16 μg at 72 h, respectively. Of the total dose, 5.35% of loureirin A and 5.46% of loureirin B were excreted from feces at 60 h. The cumulative amounts of loureirin A and loureirin B in rat bile reached 4.49 ± 0.98 and 5.11 ± 0.83 μg, respectively, at 36 h after dosing, accounting for 0.054% and 0.056% of the total dose. Figure Fragmentation pathways of loureirin A (a) and loureirinB (b)

Keywords: HPLC-ESI-MS; Loureirin A; Loureirin B; Excretion; Longxuejie

Preconcentration and sensitive determination of hexabromocyclododecane diastereomers in environmental water samples using solid phase extraction with bamboo charcoal cartridge prior to rapid resolution liquid chromatography–electrospray tandem mass spectrometry by Ru-Song Zhao; Cong Hu; Jia-Bin Zhou; Jin-Peng Yuan; Shan-Shan Wang; Xia Wang (pp. 1189-1195).

In this paper, a simple and cheap method for the simultaneous preconcentration and sensitive determination of three hexabromocyclododecane (HBCD) diastereomers (α-, β-, and γ-HBCD) in environmental water samples has been developed. It was based on solid phase extraction (SPE) and rapid resolution liquid chromatography–electrospray tandem mass spectrometry. Bamboo charcoal, one kind of cheap material, was investigated and used as SPE adsorbent for the enrichment and determination of HBCD diastereomers. Related important parameters affecting extraction efficiencies, including type and volume of eluant, amount of sorbent, sample pH, flow rate, and sample volume, were investigated and optimized in detail. Under the optimum conditions, experimental data exhibited excellent linear relationships between peak area and concentrations over the range 0.1–10 μg L⁻¹. The limits of detection and precision were in the range of 0.005–0.015 μg L⁻¹ and 4.59–7.47%, respectively. The proposed method has been successfully applied for the trace analysis of HBCD diastereomers in real-world environmental water samples. Figure Determination of HBCD diastereomers in environmental water samples

Keywords: Bamboo charcoal; Solid phase extraction; Hexabromocyclododecane; Rapid resolution liquid chromatography–electrospray tandem mass spectrometry

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Analytical and Bioanalytical Chemistry (v.400, #4)