Towards Surface Plasmon Resonance biosensing combined with bioaffinity-assisted nano HILIC Liquid Chromatography Time-of-flight Mass Spectrometry identification of Paralytic Shellfish Poisons

The potential for coupling technologies to deliver new, improved forms of bioanalysis is still in its infancy. We review a number of examples in which coupling has been successful, with special emphasis on combining surface-plasmon-resonance biosensors with mass spectrometry. We give an overview of current progress towards combining biosensor-based bioanalysis with chemical analysis for confirmation of paralytic shellfish poisons that are marine toxins. This comprehensive approach could be an alternative to the official methods currently used (e.g., animal testing and high-performance liquid chromatography with fluorescence detection) and could serve as a model for many more such applications. (C) 2009 Elsevier Ltd. All rights reserved.

Development of a high-throughput enzyme-linked immunosorbent assay for the routine detection of the carcinogen acrylamide in food, via rapid derivatisation pre-analysis

The spontaneous formation of the neurotoxic carcinogen acrylamide in a wide range of cooked foods has recently been discovered. These foods include bread and other bakery products, crisps, chips, breakfast cereals, and coffee. To date, the diminutive size of acrylamide (71.08Da) has prevented the development of screening immunoassays for this chemical. In this study, a polyclonal antibody capable of binding the carcinogen was produced by the synthesis of an immunogen comprising acrylamide derivatised with 3-mercaptobenzoic acid (3-MBA), and its conjugation to the carrier protein bovine thyroglobulin. Antiserum from the immunised rabbit was harvested and fully characterised. it displayed no binding affinity for acrylamide or 3-MBA but had a high affinity for 3-MBA-derivitised acrylamide. The antisera produced was utilised in the development of an ELISA based detection system for acrylamide. Spiked water samples were assayed for acrylamide content using a previously published extraction method validated for coffee, crispbread, potato, milk chocolate and potato crisp matrices. Extracted acrylamide was then subjected to a rapid 1-h derivatisation with 3-MBA, pre-analysis. The ELISA was shown to have a high specificity for acrylamide, with a limit of detection in water samples of 65.7 mu g kg(-1), i.e. potentially suitable for acrylamide detection in a wide range of food commodities. Future development of this assay will increase sensitivity further. This is the first report of an immunoassay capable of detecting the carcinogen, as its small size has necessitated current analytical detection via expensive, slower, physico-chemical techniques such as Gas or Liquid Chromatography coupled to Mass Spectrometry. (c) 2007 Elsevier B.V. All rights reserved.

Effective monitoring for ractopamine residues in samples of animal origin by SPR biosensor and mass spectrometry

Ractopamine (RCT) is a member of the beta-2-agonist (beta-agonist) family. It is licensed for use as an animal growth promoter in more than 20 countries worldwide, including the United States and Canada, but is either not licensed or prohibited by over 150 others, including those within the European Union. The issue of the use of RCT in livestock bound for human consumption has risen to prominence recently following the decision by The People's Republic of China to ban the import of pork from a number of processing plants after finding traces of RCT in shipments from the U.S.A.

Assessment of specific binding proteins suitable for the detection of paralytic shellfish poisons using optical biosensor technology

Paralytic shellfish poisoning (PSP) toxin monitoring in shellfish is currently performed using the internationally accredited AOAC mouse bioassay. Due to ethical and performance-related issues associated with this bioassay, the European Commission has recently published directives extending procedures that may be used for official PSP control. The feasibility of using a surface plasmon resonance optical biosensor to detect PSP toxins in shellfish tissue below regulatory levels was examined. Three different PSP toxin protein binders were investigated: a sodium channel receptor (SCR) preparation derived from rat brains, a monoclonal antibody (GT13-A) raised to gonyautoxin 2/3, and a rabbit polyclonal antibody (R895) raised to saxitoxin (STX). Inhibition assay formats were used throughout. Immobilization of STX to the biosensor chip surface was achieved via amino-coupling. Specific binding and inhibition of binding to this surface was achieved using all proteins tested. For STX calibration curves, 0 - 1000 ng/mL, IC50 values for each binder were as follows: SCR 8.11 ng/mL; GT13-A 5.77 ng/mL; and R895 1.56 ng/mL. Each binder demonstrated a different cross-reactivity profile against a range of STX analogues. R895 delivered a profile that was most likely to detect the widest range of PSP toxins at or below the internationally adopted regulatory limits.

Development of a novel immunobiosensor method for the rapid detection of okadaic acid contamination in shellfish extracts

The mouse bioassay is the methodology that is most widely used to detect okadaic acid (OA) in shellfish samples. This is one of the best-known toxins, and it belongs to the family of marine biotoxins referred to as the diarrhetic shellfish poisons (DSP). Due to animal welfare concerns, alternative methods of toxin detection are being sought. A rapid and specific biosensor immunoassay method was developed and validated for the detection of OA. An optical sensor instrument based on the surface plasmon resonance (SPR) phenomenon was utilised. A polyclonal antibody to OA was raised against OA-bovine thyroglobulin conjugate and OA-N-hydroxy succinimide ester was immobilised onto an amine sensor chip surface. The assay parameters selected for the analysis of the samples were: antibody dilution, 1/750; ratio of antibody to standard, 1:1; volume of sample injected, 25 mu l min(-1); flow rate, 25 mu l min(-1). An assay action limit of 126 ng g(-1) was established by analysing of 20 shellfish samples spiked with OA at the critical concentration of 160 ng g(-1), which is the action limit established by the European Union (EU). At this concentration of OA, the assay delivered coefficient of variations (CVs) of

Immunobiosensor detection of domoic acid as a screening test in bivalve molluscs: Comparison with liquid chromatography-based analysis

A rapid and sensitive immuno-based screening method was developed to detect domoic acid (DA) present in extracts of shellfish species using a surface plasmon resonance-based optical biosensor. A rabbit polyclonal antibody raised against DA was mixed with standard or sample extracts and allowed to interact with DA immobilized onto a sensor chip surface. The characterization of the antibody strongly suggested high cross-reactivity with DA and important isomers of the toxin. The binding of this antibody to the sensor chip surface was inhibited in the presence of DA in either standard solutions or sample extracts. The DA chip surface proved to be highly stable, achieving approximately 800 analyses per chip without any loss of surface activity. A single analytical cycle (sample injection, chip regeneration, and system wash) took 10 min to complete. Sample analysis (scallops, mussels, cockles, oysters) was achieved by simple extraction with methanol. These extracts were then filtered and diluted before analysis. Detection limits in the ng/g range were achieved by the assay; however, the assay parameters chosen allowed the test to be performed most accurately at the European Union's official action limit for DA of 20 mu g/g. At this concentration, intra- and interassay variations were measured for a range of shellfish species and ranged from 4.5 to 7.4% and 2.3 to 9.7%, respectively.

Rapid screening for monensin residues in poultry plasma by a dry reagent dissociation enhanced lanthanide fluoroimmunoassay.

Monensin, a carboxylic acid ionophore, is commonly fed to poultry to control coccidiosis. A method for rapid analysis of unextracted poultry plasma samples has been developed based on a novel immunoassay format: one-step all-in-one dry reagent time resolved fluorimetry. All assay specific components were pre-dried onto microtitration plate wells. Only addition of the serum sample diluted in assay buffer was required to perform analysis. Results were available one hour after sample addition. The limit of detection (mean + 3s) of the assay calculated from the analysis of 23 known negative samples was 14.2 ng ml(-1). Intra- and inter-assay RSD were determined as 15.2 and 7.4%, respectively, using a plasma sample fortified with 50 ng ml(-1) monensin. Eight broiler chickens were fed monensin at a dose rate of 120 mg kg(-1) feed for one week, blood sampled then slaughtered without drug withdrawal. Plasma monensin concentrations, as determined by the fluoroimmunoassay ranged from 101-297 ng ml(-1). This compared with monensin liver concentrations, determined by LC-MS, which ranged fi om 13-41 ng g(-1). The fluoroimmunoassay described is extremely user friendly, gives particularly rapid results and is suitable for the detection and quantification of plasma monensin residues. Data from medicated poultry suggest that analysis of plasma may be useful in predicting the extent of monensin liver residues.

Studies on the persistence of estradiol benzoate and nortestosterone decanoate in hair of cattle following treatment with growth promoters, determined by ultra-high-performance liquid chromatography-tandem mass spectrometry

Measurement of steroid esters in bovine hair samples, using sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS), provides a powerful tool for identifying animals treated illicitly with growth promoters. The successful application of such testing requires appropriate sampling of hair from treated animals. This paper describes the results of hair analysis by LC-MS/MS for two animal studies in which animals were treated with estradiol-3-benzoate and nortestosterone decanoate. The results from the first animal study indicate that animals treated with these anabolic steroids may not always be identified from analysis of hair samples; positive test results occur sporadically and only for some of the treated animals. The results from the second animal study identify conditions attaching to positive hair samples, such as, that concentrations of steroid esters in hair are related to distance of sampling from point of injection and to time post-treatment, that concentrations of steroid esters in hair are related to dose given to the animal but that this relationship may vary over time post-treatment, and that steroid esters may be measured in regrowth hair taken some weeks after treatment. Steroid esters are determined along the length of the hair, confirming that accumulation of steroid esters into hair occurs from various sources, including blood, sweat and sebum. The reported research provides some useful insights into the mechanisms governing the persistence of steroid esters in bovine hair following illicit treatment with growth promoters. (C) 2009 Elsevier B.V. All rights reserved.

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.

Biosensor-based detection of reduced sex hormone-binding globulin binding capacities in response to growth-promoter administrations

Growth-promoting agents are illicitly used during animal rearing processes and the detection of their use is limited by new compounds and dosing practices that limit the efficiency of current testing which is based on residue analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and gas chromatography–mass spectrometry (GC–MS) methodology. An alternative approach is to use indirect biological evidence as a screening tool to identify growth-promoter treated animals thus improving the effectiveness of residue testing through the targeted sampling of these animals. Sex hormone-binding globulin (SHBG) is a glycoprotein which binds and controls the levels of sex-hormones within the circulation. Using a biosensor assay based on measurement of binding to an immobilised 1a-dihydrotestosterone (1a-DHT) derivative, reduced SHBG binding capacities were detected in growth-promoter treated animals. During the course of a veal treatment regime based on repeated oestradiol benzoate, nortestosterone decanoate and dexamethasone administrations, treated male and female calves were shown to have significantly lower SHBG capacities. To assess the effectiveness of using SHBG binding capacities as a biomarker of treatment and to investigate the role of individual growth-promoter components to the SHBG capacity lowering effects, adult heifer animals were subjected to repeated doses of nortestosterone decanoate. These animals also demonstrated a reduction in SHBG capacity levels at Day 39 of the study, in contrast to oestradiol benzoate treated adult steers who were found to have unaltered levels. These findings suggest that the measurement of SHBG binding capacities using a biosensor assay has potential in the identification of illegally treated animals, particularly those exposed to androgens.

Modelling the effect of water immersion thermal processing on polyacetylene levels and instrumental colour of carrot disks

C₁₇ polyacetylenes are a group of bioactive compounds present in carrots which have recently gained scientific attention due to their cytotoxicity against cancer cells. In common with many bioactive compounds, their levels may be influenced by thermal processes, such as boiling or water immersion. This study investigated the effect of a number of water immersion time/temperature combinations on concentrations of these compounds and attempted to model the changes. Carrot samples were thermally treated by heating in water at temperatures from 50–100 °C and holding times of 2–60 min. Following heating, levels of falcarinol (FaOH), falcarindiol (FaDOH), falcarindiol-3-acetate (FaDOAc) and Hunter colour parameters (L^*, a^*, b^*) were determined. FaOH, FaDOH, FaDOAc levels were significantly reduced at lower temperatures (50–60 °C). In contrast, samples heated at temperatures from 70–100 °C exhibited higher levels of polyacetylenes (p < 0.05) than did raw unprocessed samples. Regression modelling was used to model the effects of temperature and holding time on the levels of the variables measured. Temperature treatment and holding time were found to significantly affect the polyacetylene content of carrot disks. Predicted models were found to be significant (p < 0.05) with high coefficients of determination (R²).

Thin film composite optical waveguides for sensor applications: a review

We review the design and fabrication of thin-film composite optical waveguides (OWG) with high refractive index for sensor applications. A highly sensitive optical sensor device has been developed on the basis of thin-film, composite OWG. The thin-film OWG was deposited onto the surface of a potassium-ion-exchanged (K+) glass OWG by sputtering or spin coating (5-9 mm wide, and with tapers at both ends). By allowing an adiabatic transition of the guided light from the secondary OWG to the thin-film OWG, the electric field of the evanescent wave at the thin film was enhanced. The attenuation of the guided light in the thin film layer was small, and the guided light intensity changed sensitively with the refractive index of the cladding layer. Our experimental results demonstrate that thin-film, composite OWG gas sensors or immunosensors are much more sensitive than sensors based on other technologies. (c) 2004 Elsevier B.V. All rights reserved.

The application of Reporter Gene Assays for the detection of endocrine disruptors in sport supplements.

The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC50 of 0.01 ng mL-1 and 0.16 ng mL-1 respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC–MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC–MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally active compounds the combination of biological and physio-chemical techniques is optimal.

The Ectocarpus genome and the independent evolution of multicellularity in brown algae

Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related(1). These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity (Fig. 1). We report the 214 million base pair (Mbp) genome sequence of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for brown algae(2-5), closely related to the kelps(6,7) (Fig. 1). Genome features such as the presence of an extended set of light-harvesting and pigment biosynthesis genes and new metabolic processes such as halide metabolism help explain the ability of this organism to cope with the highly variable tidal environment. The evolution of multicellularity in this lineage is correlated with the presence of a rich array of signal transduction genes. Of particular interest is the presence of a family of receptor kinases, as the independent evolution of related molecules has been linked with the emergence of multicellularity in both the animal and green plant lineages. The Ectocarpus genome sequence represents an important step towards developing this organism as a model species, providing the possibility to combine genomic and genetic(2) approaches to explore these and other(4,5) aspects of brown algal biology further.