Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009-2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Point-and-shoot: rapid quantitative detection methods for on-site food fraud analysis – moving out of the laboratory and into the food supply chain

Major food adulteration and contamination events occur with alarming regularity and are known to be episodic, with the question being not if but when another large-scale food safety/integrity incident will occur. Indeed, the challenges of maintaining food security are now internationally recognised. The ever increasing scale and complexity of food supply networks can lead to them becoming significantly more vulnerable to fraud and contamination, and potentially dysfunctional. This can make the task of deciding which analytical methods are more suitable to collect and analyse (bio)chemical data within complex food supply chains, at targeted points of vulnerability, that much more challenging. It is evident that those working within and associated with the food industry are seeking rapid, user-friendly methods to detect food fraud and contamination, and rapid/high-throughput screening methods for the analysis of food in general. In addition to being robust and reproducible, these methods should be portable and ideally handheld and/or remote sensor devices, that can be taken to or be positioned on/at-line at points of vulnerability along complex food supply networks and require a minimum amount of background training to acquire information rich data rapidly (ergo point-and-shoot). Here we briefly discuss a range of spectrometry and spectroscopy based approaches, many of which are commercially available, as well as other methods currently under development. We discuss a future perspective of how this range of detection methods in the growing sensor portfolio, along with developments in computational and information sciences such as predictive computing and the Internet of Things, will together form systems- and technology-based approaches that significantly reduce the areas of vulnerability to food crime within food supply chains. As food fraud is a problem of systems and therefore requires systems level solutions and thinking.

Elucidation of the electrochemical oxidation pathway of ammonia in dimethylformamide and the room temperature ionic liquid, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide

The direct electrochemical oxidation of ammonia has been examined in both the organic solvent dimethylformamide (DMF) and the room temperature ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [EMIM][N(Tf)(2)]. The corresponding voltammetric responses have been shown to be similar in each solvent with a broad oxidative wave occurring upon the introduction of ammonia to the solution and the appearance of a new reductive wave following the oxidation. The oxidative reaction process has been examined and a suitable reaction pathway has been deduced, corresponding to the formation of ammonium cations after oxidation of the ammonia. A linear response of limiting current against vol% ammonia was observed in both DMF and [EMIM][N(Tf)(2)], suggesting potential application for analytical methods.

Radiotherapy in the presence of contrast agents: a general figure of merit and its application to gold nanoparticles

Delivering sufficient dose to tumours while sparing surrounding tissue is one of the primary challenges of radiotherapy, and in common practice this is typically achieved by using highly penetrating MV photon beams and spatially shaping dose. However, there has been a recent increase in interest in the possibility of using contrast agents with high atomic number to enhance the dose deposited in tumours when used in conjunction with kV x-rays, which see a significant increase in absorption due to the heavy element's high-photoelectric cross-section at such energies. Unfortunately, the introduction of such contrast agents significantly complicates the comparison of different source types for treatment efficacy, as the dose deposited now depends very strongly on the exact composition of the spectrum, making traditional metrics such as beam quality less valuable. To address this, a 'figure of merit' is proposed, which yields a value which enables the direct comparison of different source types for tumours at different depths inside a patient. This figure of merit is evaluated for a 15 MV LINAC source and two 150 kVp sources (both of which make use of a tungsten target, one with conventional aluminium filtration, while the other uses a more aggressive thorium filter) through analytical methods as well as numerical models, considering tissue treated with a realistic concentration and uptake ratio of gold nanoparticle contrast agents (10 mg ml(-1) concentration in 'tumour' volume, 10: 1 uptake ratio). Finally, a test case of human neck phantom is considered with a similar contrast agent to compare the abstract figure to a more realistic treatment situation. Good agreement was found both between the different approaches to calculate the figure of merit, and between the figure of merit and the effectiveness in a more realistic patient scenario. Together, these observations suggest that there is the potential for contrast-enhanced kilovoltage radiation to be a useful therapeutic tool for a number of classes of tumour on dosimetric considerations alone, and they point to the need for further research in this area.

The construction of a whole-cell biosensor for phosphonoacetate, based on the LysR-like transcriptional regulator PhnR from Pseudomonas fluorescens 23F

The phnA gene that encodes the carbon-phosphorus bond cleavage enzyme phosphonoacetate hydrolase is widely distributed in the environment, suggesting that its phosphonate substrate may play a significant role in biogeochemical phosphorus cycling. Surprisingly, however, no biogenic origin for phosphonoacetate has yet been established. To facilitate the search for its natural source we have constructed a whole-cell phosphonoacetate biosensor. The gene encoding the LysR-type transcriptional activator PhnR, which controls expression of the phosphonoacetate degradative operon in Pseudomonas fluorescens 23F, was inserted in the broad-host-range promoter probe vector pPROBE-NT, together with the promoter region of the structural genes. Cells of Escherichia coli DH5a that contained the resultant construct, pPANT3, exhibited phosphonoacetate-dependent green fluorescent protein fluorescence in response to threshold concentrations of as little as 0.5 µM phosphonoacetate, some 100 times lower than the detection limit of currently available non-biological analytical methods; the pPANT3 biosensor construct in Pseudomonas putida KT2440 was less sensitive, although with shorter response times. From a range of other phosphonates and phosphonoacetate analogues tested, only phosphonoacetaldehyde and arsonoacetate induced green fluorescent protein fluorescence in the E. coli DH5a (pPANT3) biosensor, although at much-reduced sensitivities (50 µM phosphonoacetaldehyde and 500 µM arsonoacetate).

A competitive enzyme-linked immunosorbent assay for determination of chloramphenicol

Chloramphenicol is a broad-spectrum antibiotic shown to have specific activity against a wide variety of organisms that are causative agents of several disease conditions in domestic animals. Chloramphenicol has been banned for use in food-producing animals for its serious adverse toxic effects in humans. Due to the harmful effects of chloramphenicol residues livestock products should be free of any traces of these residues. Several analytical methods are available for chloramphenicol analysis but sensitive methods are required in order to ensure that no traces of chloramphenicol residues are present in edible animal products. In order to prevent the illegal use of chloramphenicol, regulatory control of its residues in food of animal origin is essential. A competitive enzyme-linked immunosorbent assay for chloramphenicol has been locally developed and optimized for the detection of chloramphenicol in sheep serum. In the assay, chloramphenicol in the test samples and that in chloramphenicol-horseradish peroxidase conjugate compete for antibodies raised against the drug in camels and immobilized on a microtitre plate. Tetramethylbenzidine-hydrogen peroxide (TMB/H2O2) is used as chromogen-substrate system. The assay has a detection limit of 0.1 ng/mL of serum with a high specificity for chloramphenicol. Cross-reactivity with florfenicol, thiamphenicol, penicillin, tetracyclines and sulfamethazine was not observed. The assay was able to detect chloramphenicol concentrations in normal sheep serum for at least 1 week after intramuscular injection with the drug at a dose of 25 mg/kg body weight (b.w.). The assay can be used as a screening tool for chloramphenicol use in animals.

Combining biomarker screening and mass-spectrometric analysis to detect hormone abuse in cattle

Since the introduction of the European ban on hormones in 1989, its implementation has proved to be an enormous challenge to regulatory authorities, because the great economic benefits that result from illegal misuse of growth promoters in animal production encourage their continued use. In efforts to challenge black-market trade in hormones, there have been many analytical advances. Recently, both effect-based bioanalysis for screening to target illegal misuse and improved mass-spectrometry-based confirmatory analysis have greatly increased the likelihood of detecting hormone abuse. This review outlines analytical methods currently used for detecting hormone abuse and presents advances in new approaches based on biological determinants that may complement these techniques in the future. (C) 2009 Elsevier Ltd. All rights reserved.

Recent advances in the analysis of behavioural organization and interpretation as indicators of animal welfare

While the incorporation of mathematical and engineering methods has greatly advanced in other areas of the life sciences, they have been under-utilized in the field of animal welfare. Exceptions are beginning to emerge and share a common motivation to quantify 'hidden' aspects in the structure of the behaviour of an individual, or group of animals. Such analyses have the potential to quantify behavioural markers of pain and stress and quantify abnormal behaviour objectively. This review seeks to explore the scope of such analytical methods as behavioural indicators of welfare. We outline four classes of analyses that can be used to quantify aspects of behavioural organization. The underlying principles, possible applications and limitations are described for: fractal analysis, temporal methods, social network analysis, and agent-based modelling and simulation. We hope to encourage further application of analyses of behavioural organization by highlighting potential applications in the assessment of animal welfare, and increasing awareness of the scope for the development of new mathematical methods in this area.

Synthesis and characterisation of new platinum-acetylide complexes containing diimine ligands

A new class of platinum-bipyridyl compounds has been synthesized by the dehydrohalogenative reaction of [4,4'-bis(tert-butyl)-2,2'-bipyridyl]platinum dichloride [PtCl2((t)Bu(2)bipy)] 1 with terminal alkynes HC=CR, in the presence of copper(I) iodide and diisopropylamine. The products [Pt(C=CR)(2)((t)Bu(2)bipy)] (R=C6H4NO2-p 2, C6H5 3, C6H4CH3-p 4 or SiMe3 5), have been characterised by spectroscopic and analytical methods, and a single crystal molecular structure determination has been carried out on 4. Extended Huckel molecular orbital calculations have also been carried out, and the results are used to help rationalise the voltammetric, EPR and spectroelectrochemical properties of the new compounds. These show that compounds 3, 4 and 5 undergo a one-electron bipyridyl based redox process, but that 2 has an unresolved two-electron process located on the nitro groups.

An improved immunoassay for detection of saxitoxin by surface plasmon resonance biosensors

Saxitoxin and its analogs, the causative agents of paralytic shellfish poisoning (PSP), are a worldwide threat to seafood safety. Effective monitoring of potentially contaminated fishing areas as well as screening of seafood samples is necessary to adequately protect the public. While many analytical methods exist for detecting paralytic shellfish toxins (PSTs), each technique has challenges associated with routine use. One recently developed method [1] that overcomes ethical or performance-related issues of other techniques is the surface plasmon resonance (SPR) bioassay. Notwithstanding the advantages of this method, much research remains in optimizing the sensor substrate and assay conditions to create a robust technique for rapid and sensitive measurement of PSTs. This manuscript describes a more rigorous and stable SPR inhibition immunoassay through optimization of the surface chemistry as well as determination of optimum mixture ratios and mixing times. The final system provides rapid substrate formation (18 h saxitoxin conjugation with low reagent consumption), contains a reference channel for each assay, and is capable of triplicate measurements in a single run with detection limits well below the regulatory action level. Published by Elsevier B.V.

Detection of 3-amino-2-oxazolidinone (AOZ), a tissue-bound metabolite of the nitrofuran furazolidone, in prawn tissue by enzyme immunoassay

Furazolidone, a nitrofuran antibiotic, is banned from use in food animal production within the European Union. Increasingly, compliance with this ban is monitored by use of analytical methods to detect a stable tissue-bound metabolite, 3-amino-2-oxazolidinone (AOZ). Widespread use of furazolidone in poultry and prawns imported into Europe highlighted the urgent need for development of nitrofuran immunoassay screening tests. The first enzyme-linked immunoabsorbant assay for detection of AOZ residues in prawns (shrimps) is now described. Prawn samples were derivatized with o-nitrobenzaldehyde, extracted into ethyl acetate, washed with hexane and applied to a competitive enzyme immunoassay based on a rabbit polyclonal antiserum. Assay limit of detection (LOD) (mean+3 s) calculated from the analysis of 20 known negative cold and warm water prawn samples was 0.1 mug kg(-1). Intra- and interassay relative standard deviations were determined as 18.8 and 38.2%, respectively, using a negative prawn fortified at 0.7 mug kg(-1). The detection capability (CCbeta), defined as the concentration of AOZ at which 20 different fortified samples yielded results above the LOD, was achieved at fortification between 0.4 and 0.7 mug kg(-1). Incurred prawn samples (n=8) confirmed by liquid chromatography coupled with tandem mass spectrometry detection to contain AOZ concentrations between 0.4 and 12.7 mug kg(-1) were all screened positive by this enzyme-linked immunoabsorbant assay. Further data are presented and discussed with regard to calculating assay LOD based on accepting a 5% false-positive rate with representative negative prawn samples. Such an acceptance improves the sensitivity of an ELISA and in this case permitted an LOD of 0.05 mug kg(-1) and a CCbeta of below 0.4 mug kg(-1).

Monoclonal antibody-based ELISA for the quantification of nitrofuran metabolite 3-amino-2-oxazolidinone in tissues using a simplified sample preparation

A sensitive and specific monoclonal ELISA for the determination of tissue bound furazolidone metabolite 3-amino-2-oxazolidinone (AOZ) is described. The procedure enables the detection of AOZ in matrix supernatant after homogenisation, protease treatment, acid hydrolysis and derivatisation of AOZ released from the tissue by o-nitrobenzaldehyde. The formed p-nitrophenyl 3-amino-2-oxazolidinone (NPAOZ) is determined by ELISA calibrated with matrix-matched standards in the concentration range of 0.05-5.0 mu g l(-1). The assay was validated according to criteria set down by Commission Decision 2002/657/EC for the performance and validation of analytical methods for chemical residues. Detection capability, set on the basis of acceptance of no false negative results, was 0.4 mu g kg(-1) for shrimp, poultry, beef and pork muscle. This sensitivity approaches the established confirmatory LC-MS/MS able to quantify tissue-bound AOZ at levels as low as 0.3 mu g kg(-1). An excellent correlation of results obtained by ELISA and LC/MS-MS within the concentration range 0-32.1 mu g kg(-1) was found in the naturally contaminated shrimp samples (r = 0.999, n = 8). A similar con-elation was found for the incurred poultry samples within the concentration range of 0-10.5 mu g kg(-1) (r = 0.99, n = 8). (c) 2005 Elsevier B.V All rights reserved.

Molecularly imprinted polymers for the extraction of imiquimod from biological samples using a template analogue strategy

Molecularly Imprinted Polymers (MIPs) against imiquimod, a highly potent immune response modifier used in the treatment of skin cancer, were synthesised using a template analogue strategy and were compared with imprints of the drug itself. An investigation of the complexation between the functional monomer and the template analogue revealed an association constant of 1,376 ± 122 M-1, significantly higher than previously reported values for similar systems. The binding characteristics of the synthesised imprinted polymers were evaluated and extremely strong binding for imiquimod was observed while imprinting factors as high as 17 were calculated. When applied as sorbents in solid-phase extraction of imiquimod from aqueous, urine and blood serum samples, clean extracts and recoveries up to 95% were achieved, and it is concluded that while imiquimod imprints exhibited higher capacity for the drug, template analogue imprints are more selective. The results obtained suggest potential applications of imiquimod imprints as sorbents in rapid extraction and monitoring of undesirable systemic release of the drug.