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Analytical and Bioanalytical Chemistry (v.402, #10)

Biomimetic recognition elements for sensing applications by María Cruz Moreno-Bondi (pp. 3019-3020).

received a Ph.D. in analytical chemistry at Complutense University of Madrid (Spain) in 1990. She was promoted to full Professor at the Department of Analytical Chemistry of Complutense University in 2008. She received the Young Researcher’s Award from the Spanish Society of Analytical Chemistry in 1993 and the Research Award in Analytical Chemistry from the Royal Spanish Society of Chemistry in 2010. Her main research interests include the development of optical chemical sensors and biosensors based on fluorescence measurements, the synthesis of biomimetic recognition elements for sensing and separation purposes, and the application of these to environmental and food analysis

Molecularly imprinted polymers as biomimetic catalysts by Marina Resmini (pp. 3021-3026).

The quest for synthetic biomimetic catalysts able to complement the activity of enzymes has attracted substantial research efforts, and the molecular imprinting approach is one of the attractive techniques that are currently being investigated. In the last 3 years, there has been considerable interest in studying in greater detail the parameters that control and influence the catalytic activity of imprinted polymers and applying molecular imprinting to a wider range of polymeric matrices. This article reports on some of the interesting examples available in the literature regarding the use of metal-containing polymers, microgels and nanogels and thermoresponsive polymers.

Keywords: Molecular imprinting; Imprinted polymers; Catalysis; Biomimetics; Enzyme mimics

Recombinant antibodies and their use in biosensors by Xiangqun Zeng; Zhihong Shen; Ray Mernaugh (pp. 3027-3038).

Inexpensive, noninvasive immunoassays can be used to quickly detect disease in humans. Immunoassay sensitivity and specificity are decidedly dependent upon high-affinity, antigen-specific antibodies. Antibodies are produced biologically. As such, antibody quality and suitability for use in immunoassays cannot be readily determined or controlled by human intervention. However, the process through which high-quality antibodies can be obtained has been shortened and streamlined by use of genetic engineering and recombinant antibody techniques. Antibodies that traditionally take several months or more to produce when animals are used can now be developed in a few weeks as recombinant antibodies produced in bacteria, yeast, or other cell types. Typically most immunoassays use two or more antibodies or antibody fragments to detect antigens that are indicators of disease. However, a label-free biosensor, for example, a quartz-crystal microbalance (QCM) needs one antibody only. As such, the cost and time needed to design and develop an immunoassay can be substantially reduced if recombinant antibodies and biosensors are used rather than traditional antibody and assay (e.g. enzyme-linked immunosorbant assay, ELISA) methods. Unlike traditional antibodies, recombinant antibodies can be genetically engineered to self-assemble on biosensor surfaces, at high density, and correctly oriented to enhance antigen-binding activity and to increase assay sensitivity, specificity, and stability. Additionally, biosensor surface chemistry and physical and electronic properties can be modified to further increase immunoassay performance above and beyond that obtained by use of traditional methods. This review describes some of the techniques investigators have used to develop highly specific and sensitive, recombinant antibody-based biosensors for detection of antigens in simple or complex biological samples. Figure 1 Recombinant antibody and its common surface coupling strategies

Keywords: Recombinant antibody; Biosensors; Piezoimmunosensors; QCM

Reagentless fluorescent biosensors based on proteins for continuous monitoring systems by Javier Galbán; Isabel Sanz-Vicente; Estefania Ortega; Melisa del Barrio; Susana de Marcos (pp. 3039-3054).

There is a lack of commercially available efficient and autonomous systems capable of continuous monitoring of (bio)chemical data for clinical, environmental, food, or industrial samples. The weakest link in the design of these systems is the (bio)chemical receptor (bCR). The bCR should have transducer ability, the recognition event should be a single reaction, and the bCR should be easily regenerated. Transport proteins and enzymes are well placed as bCR for optical continuous monitoring systems (OCMS). In this paper we review quantitative aspects and the main transducer strategies which have been developed for transport proteins, using periplasmic binding proteins (linking an environmentally sensitive fluorophore or FRET between two fluorophores) and concanavalin A (competitive reversible assays) as representative examples. Efficient immobilization systems and implementation in OCMS are also reviewed. Some kinds of enzymes can fulfil the necessary requirements to be appropriate bCR. Strategies using flavoenzymes chemically modified with fluorophores can be successfully implemented in OCMS and they are, in our opinion, the most appropriate option.

Keywords: Biosensors; Fluorescence/luminescence; Optical sensors; Organic compounds/trace organic compounds; Clinical/biomedical analysis

Short peptides as biosensor transducers by Silvia Pavan; Federico Berti (pp. 3055-3070).

This review deals with short peptides (up to 50 amino acids) as biomimetic active recognition elements in sensing systems. Peptide-based sensors have been developed in recent years according to different strategies. Synthetic peptides have been designed on the basis of known interactions between single or a few amino acids and targets, with attention being paid to the presence of peptide motifs known to allow intermolecular self-organization of the sensing peptides over the sensor surface. Sensitive and sophisticated sensors have been obtained in this way, but the use of designed peptides is limited by severe difficulties in their in silico design. Short peptides from random phage display have been selected in a random way from large, unfocussed, and often preexisting and commercially available phage display libraries, with no design elements. Such peptides often perform better than antibodies, but they are difficult to select when the target is a small molecule because of the need to immobilize it with considerable modifications of its structure. Artificial, miniaturized receptors have been obtained from the reduction of the known sequence of a natural receptor down to a synthesizable and yet stable one. Alternatively, binding sites have been created over a designed, stable peptide scaffold. Short peptides have also been used as active elements for the detection of their own natural receptors: pathogenic bacteria have been detected with antimicrobial and cell-penetrating peptides, but key challenges such as detection of bacteria in real samples, improved sensitivity, and improved selectivity have to be faced. Peptide substrates have been conjugated to fluorescent quantum dots to obtain disposable sensors for protease activity with high sensitivity. Ferrocene–peptide conjugates have been used for electrochemical sensing of protease activity.

Keywords: Amino acids; Peptides, Biosensors, Electrochemical sensors; Mass sensitive sensors, Fluorescence; Luminescence, Optical sensors

Applications of peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) in biosensor development by Carlos Briones; Miguel Moreno (pp. 3071-3089).

Nucleic acid biosensors have a growing number of applications in genetics and biomedicine. This contribution is a critical review of the current state of the art concerning the use of nucleic acid analogues, in particular peptide nucleic acids (PNA) and locked nucleic acids (LNA), for the development of high-performance affinity biosensors. Both PNA and LNA have outstanding affinity for natural nucleic acids, and the destabilizing effect of base mismatches in PNA- or LNA-containing heterodimers is much higher than in double-stranded DNA or RNA. Therefore, PNA- and LNA-based biosensors have unprecedented sensitivity and specificity, with special applicability in DNA genotyping. Herein, the most relevant PNA- and LNA-based biosensors are presented, and their advantages and their current limitations are discussed. Some of the reviewed technology, while promising, still needs to bridge the gap between experimental status and the harder reality of biotechnological or biomedical applications.

Keywords: Nucleic acid analogue; DNA; Hybridization; SNP; Microarray; Self-assembled monolayer

Recent trends in molecular beacon design and applications by Kewei Huang; Angel A. Martí (pp. 3091-3102).

A molecular beacon (MB) is a hairpin-structured oligonucleotide probe containing a photoluminescent species (PLS) and a quencher at different ends of the strand. In a recognition and detection process, the hybridization of MBs with target DNA sequences restores the strong photoluminescence, which is quenched before hybridization. Making better MBs involves reducing the background photoluminescence and increasing the brightness of the PLS, which therefore involves the development of new PLS and quenchers, as well as innovative PLS–quencher systems. Heavy-metal complexes, nanocrystals, pyrene compounds, and other materials with excellent photophysical properties have been applied as PLS of MBs. Nanoparticles, nanowires, graphene, metal films, and many other media have also been introduced to quench photoluminescence. On the basis of their high specificity, selectivity, and sensitivity, MBs are developed as a general platform for sensing, producing, and carrying molecules other than oligonucleotides.

Keywords: Molecular beacon; Photoluminescence; Metal complex; Quantum dot; Pyrene; Nanoquencher

Electrochemical nanomaterial-based nucleic acid aptasensors by Ilaria Palchetti; Marco Mascini (pp. 3103-3114).

Recent progress in the development of electrochemical nanomaterial–aptamer-based biosensors is summarized. Aptamers are nucleic acid ligands that can be generated against amino acids, drugs, proteins, and other molecules. They are isolated from a large random library of synthetic nucleic acids by an iterative process of binding, separation, and amplification, called systematic evolution of ligands by exponential enrichment (SELEX). In this review, different methods of integrating aptamers with different nanomaterials and nanoparticles for electrochemical biosensing application are described.

Keywords: Nucleic acid aptamers; Nanomaterials; Nanoparticles; Electrochemical biosensors

Fluorescent hybridization probes for nucleic acid detection by Jia Guo; Jingyue Ju; Nicholas J. Turro (pp. 3115-3125).

Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent hybridization probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary probes (BPs) are two very important hybridization probes that are designed based on well-established photophysical principles. These probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various hybridization probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.

Keywords: Molecular beacon; Binary probes; Fluorescence; FRET; Oligonucleotide; Signal-to-noise ratio

Pathogen detection using engineered bacteriophages by Abby E. Smartt; Tingting Xu; Patricia Jegier; Jessica J. Carswell; Samuel A. Blount; Gary S. Sayler; Steven Ripp (pp. 3127-3146).

Bacteriophages, or phages, are bacterial viruses that can infect a broad or narrow range of host organisms. Knowing the host range of a phage allows it to be exploited in targeting various pathogens. Applying phages for the identification of microorganisms related to food and waterborne pathogens and pathogens of clinical significance to humans and animals has a long history, and there has to some extent been a recent revival in these applications as phages have become more extensively integrated into novel detection, identification, and monitoring technologies. Biotechnological and genetic engineering strategies applied to phages are responsible for some of these new methods, but even natural unmodified phages are widely applicable when paired with appropriate innovative detector platforms. This review highlights the use of phages as pathogen detector interfaces to provide the reader with an up-to-date inventory of phage-based biodetection strategies.

Keywords: Bacteriophage; Bioreporter; Biosensor; Pathogen; Phage

Engineered cells as biosensing systems in biomedical analysis by Nilesh Raut; Gregory O’Connor; Patrizia Pasini; Sylvia Daunert (pp. 3147-3159).

Over the past two decades there have been great advances in biotechnology, including use of nucleic acids, proteins, and whole cells to develop a variety of molecular analytical tools for diagnostic, screening, and pharmaceutical applications. Through manipulation of bacterial plasmids and genomes, bacterial whole-cell sensing systems have been engineered that can serve as novel methods for analyte detection and characterization, and as more efficient and cost-effective alternatives to traditional analytical techniques. Bacterial cell-based sensing systems are typically sensitive, specific and selective, rapid, easy to use, low-cost, and amenable to multiplexing, high-throughput, and miniaturization for incorporation into portable devices. This critical review is intended to provide an overview of available bacterial whole-cell sensing systems for assessment of a variety of clinically relevant analytes. Specifically, we examine whole-cell sensing systems for detection of bacterial quorum sensing molecules, organic and inorganic toxic compounds, and drugs, and for screening of antibacterial compounds for identification of their mechanisms of action. Methods used in the design and development of whole-cell sensing systems are also reviewed.

Keywords: Whole-cell sensing system; Biomedical analysis; Quorum sensing molecules; Mercury; Hydroxylated polychlorinated biphenyls; Cytarabine; Antibiotics

Carbohydrate–protein interactions and their biosensing applications by Xiangqun Zeng; Cesar A. S. Andrade; Maria D. L. Oliveira; Xue-Long Sun (pp. 3161-3176).

Carbohydrate recognition is clearly present throughout nature, playing a major role in the initial attachment of one biological entity to another. The important question is whether these prevalent interactions could provide a real suitable alternative to the use of antibodies or nucleic acid for detection and identification. Currently, examples of carbohydrates being employed in biological detection systems are limited. The challenges of using carbohydrate recognition for detection mainly come from the weak affinity of carbohydrate–protein interactions, the lack of versatile carbohydrate scaffolds with well-defined structures, and the less developed high-information-content, real-time, and label-free assay technology. In this review, we focus on discussing the characteristics of carbohydrate–protein interactions in nature and the methods for carbohydrate immobilization based on surface coupling chemistry in terms of their general applicability for developing carbohydrate- and lectin-based label-free sensors. Furthermore, examples of innovative design of multivalent carbohydrate–protein interactions for sensor applications are given. We limit our review to show the feasibility of carbohydrate and lectin as recognition elements for label-free sensor development in several representative cases to formulate a flexible platform for their use as recognition elements for real-world biosensor applications. Figure Multivalent protein–carbohydrate interactions at the cell surface (left) and development of a biosensor using carbohydrates (right)

Keywords: Carbohydrate–protein interactions; Biosensors; Carbohydrate immobilization; Lectins

Electrochemically synthesized polymers in molecular imprinting for chemical sensing by Piyush S. Sharma; Agnieszka Pietrzyk-Le; Francis D’Souza; Wlodzimierz Kutner (pp. 3177-3204).

This critical review describes a class of polymers prepared by electrochemical polymerization that employs the concept of molecular imprinting for chemical sensing. The principal focus is on both conducting and nonconducting polymers prepared by electropolymerization of electroactive functional monomers, such as pristine and derivatized pyrrole, aminophenylboronic acid, thiophene, porphyrin, aniline, phenylenediamine, phenol, and thiophenol. A critical evaluation of the literature on electrosynthesized molecularly imprinted polymers (MIPs) applied as recognition elements of chemical sensors is presented. The aim of this review is to highlight recent achievements in analytical applications of these MIPs, including present strategies of determination of different analytes as well as identification and solutions for problems encountered.

Keywords: Electrochemically synthesized polymer; Electronically conducting polymer; Molecular imprinting; Chemical sensor; Molecular recognition

Single-molecule atomic force microscopy on live cells compares aptamer and antibody rupture forces by Meghan B. O’Donoghue; Xiaoli Shi; Xiaohong Fang; Weihong Tan (pp. 3205-3209).

Some researchers have questioned whether synthetic aptamers bind as robustly as natural antibodies. To address this issue, we used single-molecule atomic force microscopy to measure the rupture force between a protein and both its aptamer and its antibody. The rupture force on live cell membranes between the aptamer and protein was 46 ± 26 pN; the force with the antibody was 68 ± 33 pN, we conclude that the binding forces are about equal.

Keywords: PTK7; sgc8; Aptamer; Antibody; Rupture force; Atomic force microscopy

Biosensor for on-line fluorescent detection of trifluoroperazine based on genetically modified calmodulin by Martin González-Andrade; Elena Benito-Peña; Rachel Mata; Maria C. Moreno-Bondi (pp. 3211-3218).

This paper describes the development of a novel on-line biosensor based on a fluorescently labeled human calmodulin (CaM), hCaM M124C-mBBr, immobilized on controlled-pore glass (CPG), for the analysis of trifluoroperazine (TFP); a phenothiazine drug in human urine samples. The device was automated by packing hCaM M124C-mBBr-CPG in a continuous-flow microcell connected to a monitoring system, composed of a bifurcated optical fiber coupled to a spectrofluorometer. Operating parameters of the on-line biosensor (flow rate, sample injection volume, and carrier solution and buffer pH) were studied and optimized. Under the optimal conditions, the biosensor provides a detection and a quantification limit of 0.24 and 0.52 μg mL⁻¹, respectively, and a dynamic range from 0.52 to 61.05 μg mL⁻¹ TFP (n = 5, correlation coefficient 0.998). The response time (t ₁₀₀) was shorter than 42 s (recovery time <4.5 min) and reproducibility and repeatability of the TFP measurements, within the linear response range, were lower than 1.4 and 2.7%, respectively. The device was successfully applied to the analysis of TFP in spiked human urine samples with recoveries ranging between 97 and 101% and with RSDs lower than 5.9%. Figure Artistic depiction of the on-line biosensor working principle for trifluoroperazine analysis in urine samples.

Keywords: On-line biosensor; Fluorescence; CaM-CPG; Trifluoroperazine; Biomimetic element

Specific peptides as alternative to antibody ligands for biomagnetic separation of Clostridium tyrobutyricum spores by Maria Lavilla; Maria Moros; Sara Puertas; Valeria Grazú; María Dolores Pérez; Miguel Calvo; Jesus M. de la Fuente; Lourdes Sánchez (pp. 3219-3226).

Nowadays, the reference method for the detection of Clostridium tyrobutyricum in milk is the most-probable-number method, a very time-consuming and non-specific method. In this work, the suitability of the use of superparamagnetic beads coated with specific antibodies and peptides for bioseparation and concentration of spores of C. tyrobutyricum has been assessed. Peptide or antibody functionalized nanoparticles were able to specifically bind C. tyrobutyricum spores and concentrate them up to detectable levels. Moreover, several factors, such as particle size (200 nm and 1 μm), particle derivatization (aminated and carboxylated beads), coating method, and type of ligand have been studied in order to establish the most appropriate conditions for spore separation. Results show that concentration of spore is favored by a smaller bead size due to the wider surface of interaction in relation to particle volume. Antibody orientation, related to the binding method, is also critical in spore recovery. However, specific peptides seem to be a better ligand than antibodies, not only due to the higher recovery ratio of spores obtained but also due to the prolonged stability over time, allowing an optimal recovery of spores up to 3 weeks after bead coating. These results demonstrate that specific peptides bound to magnetic nanoparticles can be used instead of traditional antibodies to specifically bind C. tyrobutyricum spores being a potential basis for a rapid method to detect this bacterial target.

Keywords: Clostridium tyrobutyricum ; Spores; Magnetic separation; Phage display; Peptide; Antibodies

Investigating the effect of antibiotics on quorum sensing with whole-cell biosensing systems by Anjali K. Struss; Patrizia Pasini; Deborah Flomenhoft; Harohalli Shashidhar; Sylvia Daunert (pp. 3227-3236).

Quorum sensing (QS) allows bacteria to communicate with one another by means of QS signaling molecules and control certain behaviors in a group-based manner, including pathogenicity and biofilm formation. Bacterial gut microflora may play a role in inflammatory bowel disease pathogenesis, and antibiotics are one of the available therapeutic options for Crohn’s disease. In the present study, we employed genetically engineered bioluminescent bacterial whole-cell sensing systems as a tool to evaluate the ability of antibiotics commonly employed in the treatment of chronic inflammatory conditions to interfere with QS. We investigated the effect of ciprofloxacin, metronidazole, and tinidazole on quorum sensing. Several concentrations of individual antibiotics were allowed to interact with two different types of bacterial sensing cells, in both the presence and absence of a fixed concentration of N-acylhomoserine lactone (AHL) QS molecules. The antibiotic effect was then determined by monitoring the biosensor’s bioluminescence response. Ciprofloxacin, metronidazole, and tinidazole exhibited a dose-dependent augmentation in the response of both bacterial sensing systems, thus showing an AHL-like effect. Additionally, such an augmentation was observed, in both the presence and absence of AHL. The data obtained indicate that ciprofloxacin, metronidazole, and tinidazole may interfere with bacterial communication systems. The results suggest that these antibiotics, at the concentrations tested, may themselves act as bacterial signaling molecules. The beneficial effect of these antibiotics in the treatment of intestinal inflammation may be due, at least in part, to their effect on QS-related bacterial behavior in the gut.

Keywords: Bacterial whole-cell biosensing systems; Quorum sensing; Antibiotics; Bioluminescence; Inflammatory bowel disease

A photosynthetic biosensor with enhanced electron transfer generation realized by laser printing technology by Eleftherios Touloupakis; Christos Boutopoulos; Katia Buonasera; Ioanna Zergioti; Maria Teresa Giardi (pp. 3237-3244).

One of the limits of current electrochemical biosensors is a lack of methods providing stable and highly efficient junctions between biomaterial and solid-state devices. This paper shows how laser-induced forward transfer (LIFT) can enable efficient electron transfer from photosynthetic biomaterial immobilized on screen-printed electrodes (SPE). The ideal pattern, in terms of photocurrent signal of thylakoid droplets giving a stable response signal with a current intensity of approximately 335 ± 13 nA for a thylakoid mass of 28 ± 4 ng, was selected. It is shown that the efficiency of energy production of a photosynthetic system can be strongly enhanced by the LIFT process, as demonstrated by use of the technique to construct an efficient and sensitive photosynthesis-based biosensor for detecting herbicides at nanomolar concentrations.

Keywords: Laser printing; Biosensor; Photosynthesis; Herbicides

Optical sensors with molecularly imprinted nanospheres: a promising approach for robust and label-free detection of small molecules by Felix Kolarov; Klaus Niedergall; Monika Bach; Günter E. M. Tovar; Günter Gauglitz (pp. 3245-3252).

Molecularly imprinted nanospheres obtained by miniemulsion polymerization have been applied as the sensitive layer for label-free direct optical sensing of small molecules. Using these particles as the sensitive layer allowed for improving response times in comparison to sensors using MIP layers. As a model compound, well-characterized nanospheres imprinted against l-Boc-phenylalanine anilide (l-BFA) were chosen. For immobilization, a simple concept based on electrostatic adsorption was used, showing its applicability to different types of surfaces, leading to a good surface coverage. The sensor showed short response times, good selectivity, and high reversibility with a limit of detection down to 60 μM and a limit of quantitation of 94 μM. Furthermore, reproducibility, selectivity, and long-term stability of the sensitive layers were tested. The best results were achieved with an adsorption on aminopropylsilane layers, showing a chip-to-chip reproducibility of 22%. Furthermore, the sensors showed no loss in signal after a storage time of 1 year.

Keywords: Molecularly imprinted polymer; RIfS; Label free; Optical sensors; Miniemulsion polymerization; l-BFA

Fluorescent ion-imprinted polymers for selective Cu(II) optosensing by Silvia C. Lopes Pinheiro; Ana B. Descalzo; Ivo M. Raimundo Jr.; Guillermo Orellana; María C. Moreno-Bondi (pp. 3253-3260).

This paper describes the synthesis and characterization of a fluorescent ion-imprinted polymer (IIP) for selective determination of copper ions in aqueous samples. The IIP has been prepared using a novel functional monomer, 4-[(E)-2-(4′-methyl-2,2′-bipyridin-4-yl)vinyl]phenyl methacrylate (abbreviated as BSOMe) that has been spectroscopically characterized in methanolic solution, in the absence and in the presence of several metal ions, including Cd(II), Cu(II), Hg(II), Ni(II), Pb(II), and Zn(II). The stability constant (2.04 × 10⁸ mol⁻² l²) and stoichiometry (L₂M) of the BSOMe complex with Cu(II) were extracted thereof. Cu(II)-IIPs were prepared by radical polymerization using stoichiometric amounts of the fluorescent monomer and the template metal ion. The resulting cross-linked network did not show any leaching of the immobilized ligand allowing determination of Cu(II) in aqueous samples by fluorescence quenching measurements. Several parameters affecting optosensor performance have been optimized, including sample pH, ionic strength, or polymer regeneration for online analysis of water samples. The synthesized Cu(II)-IIP exhibits a detection limit of 0.04 μmol l⁻¹ for the determination of Cu(II) in water samples with a reproducibility of 3%, exhibiting an excellent selectivity towards the template ion over other metal ions with the same charge and close ionic radius. The IIP-based optosensor has been repeatedly used and regenerated for more than 50 cycles without a significant decrease in the luminescent properties and binding affinity of the sensing phase.

Keywords: Optical sensors; Fluorescence; MIP; Heavy metals; Copper ions

Elemental imaging and speciation in plant science by Jörg Feldmann; Eva M. Krupp (pp. 3261-3262).

is a professor at the University of Aberdeen and has held the Chair in Environmental Analytical Chemistry there since 2004. He obtained his Ph.D. in 1995 from the University of Essen (Germany), and was a Feodor Lynen Fellow (Alexander von Humboldt) at the University of British Columbia in Vancouver (Canada) before moving to Scotland in 1997 to become a lecturer at the University of Aberdeen. He is currently leading TESLA (Trace Element Speciation Laboratory Aberdeen) as part of the newly founded Marine Biodiscovery Centre. He is a member of the Advisory Boards for Environmental Chemistry and Analytical and Bioanalytical Chemistry, and has published more than 150 scientific papers—mainly on element speciation analysis in environmental and biological systems—and given more than 100 invited and plenary lectures at international conferences. The main focus of his research is on elemental speciation, with an emphasis on the development of hyphenated mass spectrometry for studies of the molecular forms of arsenic and their behaviour in biology and the environment. obtained her Ph.D. in analytical chemistry at the University of Essen, Germany, in 1999. She then moved to the CNRS for Bioinorganic Chemistry at the University of Pau, France, to work as a postdoctoral fellow in the element speciation group of O.F.X. Donard. From there she moved to the University of Aberdeen, Scotland, in 2005, and was appointed a lecturer in analytical and environmental chemistry in 2008 within TESLA (Trace Element Speciation Laboratory Aberdeen) and the Aberdeen Centre for Environmental Sustainability (ACES). She is a committee member of the Royal Society of Chemistry’s Analytical Division (Scotland). Her research is focussed on element speciation and related method developments, and currently on mercury in the environment and life in particular. She has published 45 scientific papers and given more than 20 invited lectures and workshops at international conferences and symposia.

Elemental imaging at the nanoscale: NanoSIMS and complementary techniques for element localisation in plants by Katie L. Moore; Enzo Lombi; Fang-Jie Zhao; Chris R. M. Grovenor (pp. 3263-3273).

The ability to locate and quantify elemental distributions in plants is crucial to understanding plant metabolisms, the mechanisms of uptake and transport of minerals and how plants cope with toxic elements or elemental deficiencies. High-resolution secondary ion mass spectrometry (SIMS) is emerging as an important technique for the analysis of biological material at the subcellular scale. This article reviews recent work using the CAMECA NanoSIMS to determine elemental distributions in plants. The NanoSIMS is able to map elemental distributions at high resolution, down to 50 nm, and can detect very low concentrations (milligrams per kilogram) for some elements. It is also capable of mapping almost all elements in the periodic table (from hydrogen to uranium) and can distinguish between stable isotopes, which allows the design of tracer experiments. In this review, particular focus is placed upon studying the same or similar specimens with both the NanoSIMS and a wide range of complementary techniques, showing how the advantages of each technique can be combined to provide a fuller data set to address complex scientific questions. Techniques covered include optical microscopy, synchrotron techniques, including X-ray fluorescence and X-ray absorption spectroscopy, transmission electron microscopy, electron probe microanalysis, particle-induced X-ray emission and inductively coupled plasma mass spectrometry. Some of the challenges associated with sample preparation of plant material for SIMS analysis, the artefacts and limitations of the technique and future trends are also discussed.

Keywords: Secondary ion mass spectrometry; NanoSIMS; Complementary techniques; Trace elements; X-ray spectroscopy

A review of recent developments in the speciation and location of arsenic and selenium in rice grain by Anne-Marie Carey; Enzo Lombi; Erica Donner; Martin D. de Jonge; Tracy Punshon; Brian P. Jackson; Mary Lou Guerinot; Adam H. Price; Andrew A. Meharg (pp. 3275-3286).

Rice is a staple food yet is a significant dietary source of inorganic arsenic, a class 1, nonthreshold carcinogen. Establishing the location and speciation of arsenic within the edible rice grain is essential for understanding the risk and for developing effective strategies to reduce grain arsenic concentrations. Conversely, selenium is an essential micronutrient and up to 1 billion people worldwide are selenium-deficient. Several studies have suggested that selenium supplementation can reduce the risk of some cancers, generating substantial interest in biofortifying rice. Knowledge of selenium location and speciation is important, because the anti-cancer effects of selenium depend on its speciation. Germanic acid is an arsenite/silicic acid analogue, and location of germanium may help elucidate the mechanisms of arsenite transport into grain. This review summarises recent discoveries in the location and speciation of arsenic, germanium, and selenium in rice grain using state-of-the-art mass spectrometry and synchrotron techniques, and illustrates both the importance of high-sensitivity and high-resolution techniques and the advantages of combining techniques in an integrated quantitative and spatial approach. Figure 1 Synchrotron X-ray Fluorescence microtomography images for a virtual cross section of a husked immature rice grain pulsed with 133 μM germanic acid through the excised panicle stem

Keywords: Arsenic; Selenium; Germanium; Rice grain; Speciation; Location

Functional characterisation of metal(loid) processes in planta through the integration of synchrotron techniques and plant molecular biology by Erica Donner; Tracy Punshon; Mary Lou Guerinot; Enzo Lombi (pp. 3287-3298).

Functional characterisation of the genes regulating metal(loid) homeostasis in plants is a major focus for phytoremediation, crop biofortification and food security research. Recent advances in X-ray focussing optics and fluorescence detection have greatly improved the potential to use synchrotron techniques in plant science research. With use of methods such as micro X-ray fluorescence mapping, micro computed tomography and micro X-ray absorption near edge spectroscopy, metal(loids) can be imaged in vivo in hydrated plant tissues at submicron resolution, and laterally resolved metal(loid) speciation can also be determined under physiologically relevant conditions. This article focuses on the benefits of combining molecular biology and synchrotron-based techniques. By using molecular techniques to probe the location of gene expression and protein production in combination with laterally resolved synchrotron techniques, one can effectively and efficiently assign functional information to specific genes. A review of the state of the art in this field is presented, together with examples as to how synchrotron-based methods can be combined with molecular techniques to facilitate functional characterisation of genes in planta. The article concludes with a summary of the technical challenges still remaining for synchrotron-based hard X-ray plant science research, particularly those relating to subcellular level research. Figure Elemental distribution in Arabidopsis seeds collected by synchrotron micro-XRF

Keywords: X-ray fluorescence; Tomography; Speciation; Functional genomics; Plants; Metals

Quantification of phytochelatins and their metal(loid) complexes: critical assessment of current analytical methodology by B. Alan Wood; Jörg Feldmann (pp. 3299-3309).

Whilst there are a variety of methods available for the quantification of biothiols in sample extracts, each has their own inherent advantages and limitations. The ease with which thiols readily oxidise not only hinders their quantification but also alters the speciation profile. The challenge faced by the analyst is not only to preserve the speciation of the sample, but also to select a method which allows the retrieval of the desired information. Given that sulfur is not a chromophore and that it cannot easily be monitored by ICP-MS, a number of direct and indirect methods have been developed for this purpose. In order to assess these methods, they are compared in the context of the measurement of arsenic–phytochelatin complexes in plant extracts. The inherent instability of such complexes, along with the instabilities of reduced glutathione and phytochelatin species,necessitates a rapid and sensitive analytical protocol. Whilst being a specific example, the points raised and discussed in this review will also be applicable to the quantification of biothiols and thiol–metal(loid) species in a wide range of systems other than just the analysis of arsenic–phytochelatin species in plant extracts. Figure Figure shows the schematic of a simultaneous online HPLC-ICP-MS/ESI-MS; the most sensitive direct technique for phytochelatin quantification and identification in biological extracts.

Keywords: Bioanalytical methods; Mass spectrometry/ICP-MS; Speciation; Biological samples; Sulfur

Protein fractionation and detection for metalloproteomics: challenges and approaches by James P. Barnett; David J. Scanlan; Claudia A. Blindauer (pp. 3311-3322).

At least one third of all proteins are thought to require a metal ion co-factor for their function. Recognition of the importance of metals in biological systems and major advances in analytical instrumentation and technology have led to the emergence of the new research area of metalloproteomics in recent years. Despite this progress, the experimental determination of in-vivo metal cofactors has remained challenging, because this requires elucidation of protein interactions with non-covalently bound metal ions. This critical review highlights current methodological approaches, focusing, in particular, on issues relating to the fractionation and separation of the metalloproteome, including recent experience with metalloproteomics for marine cyanobacteria in our laboratory. Metalloproteomics promises to deliver novel insights into fundamental biological processes in the future, but it is clear that further methodological advances are necessary to exploit the full potential of this emerging research area. Figure

Keywords: Metalloproteomics; Metalloprotein; 2D chromatography; IMAC; ICP–MS; Cyanobacteria

Application of elemental bioimaging using laser ablation ICP-MS in forest pathology: distribution of elements in the bark of Picea sitchensis following wounding by Magdalena Siebold; Patrick Leidich; Martina Bertini; Giuliana Deflorio; Jörg Feldmann; Eva M. Krupp; Erhard Halmschlager; Steve Woodward (pp. 3323-3331).

Element distribution in the bark of two 20-year-old clones of Picea sitchensis following wounding was studied using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Bark was sampled at 0, 3, and 43 days after wounding and analysed using a focused Nd:YAG laser (266 nm). Intensities of ¹³ C, ²⁵Mg, ²⁷Al, ³¹P, ³²S, ³⁹K, ⁴⁸Ca, ⁵⁵Mn, ⁵⁷Fe, ⁶³Cu and ⁶⁴Zn were measured by ICP-MS to study elemental distribution across the bark samples during the wound repair process. A clear accumulation of Mg, P and K at the boundary zone between the lesion and healthy tissue was detected in the wounded samples and was more distinctive at 43 than at 3 days after treatment. This zone of accumulation mapped onto the position of formation of the ligno-suberised boundary zone and differentiation of the wound periderm. These accumulations suggest major roles for Mg, P and K in the non-specific response of Sitka spruce both to wounding, possibly as co-factors to enzymes and energy utilisation. The LA-ICP-MS method developed in this work proved useful to study spatial element distribution across bark samples and has great potential for applications in other areas of plant pathology research.

Keywords: Bark; Element distribution; Element mapping; Element bio-imaging; Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS); Sitka spruce

Defence response of Sitka spruce before and after inoculation with Heterobasidion annosum: ¹H NMR fingerprinting of bark and sapwood metabolites by Giuliana Deflorio; Graham Horgan; Marcel Jaspars; Stephen Woodward (pp. 3333-3344).

Metabolite fingerprinting of Sitka spruce (Picea sitchensis) bark and sapwood was carried out by ¹H nuclear magnetic resonance after wounding and artificial inoculation with the white rot fungus Heterobasidion annosum sensu stricto. The aim was to determine whether metabolites would differ in clones showing differing levels of susceptibility to H. annosum, in the fungal as compared with the control treatment (wounding, no fungus) and the reference (healthy sample collected at 0 days), at two different locations on the host, and at different sampling times (3 and 43 days after treatment). The results suggested that different metabolic processes occur in bark and sapwood after wounding and fungal inoculation, compared with healthy samples collected before treatment: In bark, greater peaks were elicited in the aromatic region whereas, in sapwood, lower amounts of all metabolites were observed in inoculated samples, compared with healthy samples. Multivariate statistical analysis carried out with analysis of variance–principal component analysis showed highly significant effects of reference, location, and time (PC1), and significant effects of clone and fungus. Differences between clones were apparent in sapwood but not in bark and were due to peaks in the aliphatic and carbohydrate regions. Over time, in bark, there was a decrease in carbohydrate peaks, followed by an increase in aliphatic and aromatic peaks. Sapwood, by contrast, showed a decrease in all peaks, followed by an increase in carbohydrate and aliphatic peaks. Changes in carbohydrate levels were observed within the lesion compared with the more distal location in both bark and sapwood.

Keywords: Picea sitchensis ; Sitka spruce; NMR; Nuclear magnetic resonance; Metabolite fingerprinting; Bark; Sapwood; Wood decay fungi

Application of mass spectrometric techniques for the trace analysis of short-lived iodine-containing volatiles emitted by seaweed by Michael Kundel; Ute R. Thorenz; Jan H. Petersen; Ru-Jin Huang; Nicolas H. Bings; Thorsten Hoffmann (pp. 3345-3357).

Knowledge of the composition and emission rates of iodine-containing volatiles from major widespread seaweed species is important for modeling the impact of halogens on gas-phase atmospheric chemistry, new particle formation, and climate. In this work, we present the application of mass spectrometric techniques for the quantification of short-lived iodine-containing volatiles emitted by eight different seaweeds from the intertidal zone of Helgoland, Germany. A previously developed online time-of-flight aerosol mass spectrometric method was used to determine I₂ emission rates and investigate temporally resolved emission profiles. Simultaneously, iodocarbons were preconcentrated on solid adsorbent tubes and quantified offline using thermodesorption–gas chromatography-mass spectrometry. The total iodine content of the seaweeds was determined using microwave-assisted tetramethylammonium hydroxide extraction followed by inductively coupled-plasma mass spectrometry analysis. The highest total iodine content was found in the Laminariales, followed by the brown algae Ascophyllum nodosum, Fucus vesiculosus, Fucus serratus, and both red algae Chondrus crispus and Delesseria sanguinea. Laminariales were found to be the strongest I₂ emitters. Time series of the iodine release of Laminaria digitata and Laminaria hyperborea showed a strong initial I₂ emission when first exposed to air followed by an exponential decline of the release rate. For both species, I₂ emission bursts were observed. For Laminaria saccharina und F. serratus, a more continuous I₂ release profile was detected, however, F. serratus released much less I₂. A. nodosum and F. vesiculosus showed a completely different emission behavior. The I₂ emission rates of these species were slowly increasing with time during the first 1 to 2 h until a more or less stable I₂ emission rate was reached. The lowest I₂ emission rates were detected for the red algae C. crispus and D. sanguinea. Total iodocarbon emission rates showed almost the same general trend, however, the total iodocarbon emission rates were about one to two orders of magnitude lower than those of molecular iodine, demonstrating that I₂ is the major iodine containing volatile released by the investigated seaweed species. In addition, a clear dependency of iodocarbon emission from the ozone level (0–150 ppb O₃) was found for L. digitata.

Keywords: Molecular iodine; Iodocarbons; Total iodine; Marine boundary layer; ToF-AMS; TD-GC-MS; ICP-MS

Two-dimensional HPLC coupled to ICP-MS and electrospray ionisation (ESI)-MS/MS for investigating the bioavailability in vitro of arsenic species from edible seaweed by Cristina Garcia-Sartal; Sutthinun Taebunpakul; Emma Stokes; María del Carmen Barciela-Alonso; Pilar Bermejo-Barrera; Heidi Goenaga-Infante (pp. 3359-3369).

Edible seaweed consumption is a route of exposure to arsenic. However, little attention has been paid to estimate the bioaccessibility and/or bioavailability of arsenosugars in edible seaweed and their possible degradation products during gastrointestinal digestion. This work presents first use of combined inductively coupled plasma mass spectroscopy (ICP-MS) with electrospray ionization tandem mass spectrometry (ESI-MS/MS) with two-dimensional HPLC (size exclusion followed by anion exchange) to compare the qualitative and quantitative arsenosugars speciation of different edible seaweed with that of their bioavailable fraction as obtained using an in vitro gastrointestinal digestion procedure. Optimal extraction conditions for As species from four seaweed namely kombu, wakame, nori and sea lettuce were selected as a compromise between As extraction efficiency and preservation of compound identity. For most investigated samples, the use of ammonium acetate buffer as extractant and 1 h sonication in a water bath followed by HPLC-ICP-MS resulted in 40–61% of the total As to be found in the buffered aqueous extract, of which 86–110% was present as arsenosugars (glycerol sugar, phosphate sugar and sulfonate sugar for wakame and kombu and glycerol sugar and phosphate sugar for nori). The exception was sea lettuce, for which the arsenosugar fraction (glycerol sugar, phosphate sugar) only comprised 44% of the total extracted As. Interestingly, the ratio of arsenobetaine and dimethylarsinic acid to arsenosugars in sea lettuce extracts seemed higher than that for the rest of investigated samples. After in vitro gastrointestinal digestion, approximately 11–16% of the total As in the solid sample was found in the dialyzates with arsenosugars comprising 93–120% and 41% of the dialyzable As fraction for kombu, wakame, nori and sea lettuce, respectively. Moreover, the relative As species distribution in seaweed-buffered extracts and dialyzates was found to be very similar. Collection of specific fractions from the size-exclusion column to be analysed using anion-exchange HPLC-ESI-MS/MS enabled improved chromatographic selectivity, particularly for the less retained arsenosugar (glycerol sugar), facilitating confirmation of the presence of arsenosugars in seaweed extracts and dialyzates. Using this approach, the presence of arsenobetaine in sea lettuce samples was also confirmed.

Keywords: Arsenic speciation; Seaweed; Arsenic species bioavailability; Arsenosugars; Edible algae; HPLC-ICP-MS; (ESI)-MS/MS

Fractionation and identification of metalloproteins from a marine cyanobacterium by James P. Barnett; David J. Scanlan; Claudia A. Blindauer (pp. 3371-3377).

Trace metals are essential for the growth of marine cyanobacteria, being required for key cellular processes such as photosynthesis and respiration. Despite this, the metalloproteomes of marine cyanobacteria are at present only poorly defined. In this study, we have probed the major cobalt, iron, manganese, and nickel-binding proteins in the marine cyanobacterium Synechococcus sp. WH8102 by using two different fractionation approaches combined with peptide mass fingerprinting. For the identification of intact metalloproteins, multidimensional native chromatography was used to fractionate the proteome, followed by inorganic mass spectrometry to identify metal-enriched fractions. This approach led to the detection of nickel superoxide dismutase together with its predicted cofactor. We also explored the utility of immobilized metal affinity chromatography (IMAC) to isolate subpopulations of proteins that display affinity for a particular metal ion. We conclude that low-resolution 2D liquid chromatography is a viable fractionation technique to correlate relatively low-abundance metal ions with their few cellular destinations (e.g. Ni), but challenges remain for more abundant metals with multiple destinations such as iron. IMAC has been shown as a useful pre-fractionation technique to screen for proteins with metal-binding capacity, and may become a particularly valuable tool for the identification of metal-trafficking proteins.

Keywords: Cyanobacteria; Metalloproteomics; Metalloprotein; IMAC; ICP-MS

Erratum to: A review of recent developments in the speciation and location of arsenic and selenium in rice grain by Anne-Marie Carey; Enzo Lombi; Erica Donner; Martin D. de Jonge; Tracy Punshon; Brian P. Jackson; Mary Lou Guerinot; Adam H. Price; Andrew A. Meharg (pp. 3379-3379).

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Analytical and Bioanalytical Chemistry (v.402, #10)