Tailored Microarray Platform for the Detection of Marine Toxins

Currently, there are no fast in vitro broad spectrum screening bioassays for the detection of marine toxins. The aim of this study was to develop such an assay. In gene expression profiling experiments 17 marker genes were provisionally selected that were differentially regulated in human intestinal Caco-2 cells upon exposure to the lipophilic shellfish poisons azaspiracid-1 (AZA1) or dinophysis toxin-1 (DTX1). These 17 genes together with two control genes were the basis for the design of a tailored microarray platform for the detection of these marine toxins and potentially others. Five out of the 17 selected marker genes on this dedicated DNA microarray gave dear signals, whereby the resulting fingerprints could be used to detect these toxins. CEACAM1, DDIT4, and TUBB3 were up-regulated by both AZA1 and DTX1, TRIB3 was up-regulated by AZA1 only, and OSR2 by DTX1 only. Analysis by singleplex qRT-PCR revealed the up- and down-regulation of the selected RGS16 and NPPB marker genes by DTX1, that were not envisioned by the new developed dedicated array. The qRT-PCR targeting the DDIT4, RSG16 and NPPB genes thus already resulted in a specific pattern for AZA1 and DTX1 indicating that for this specific case qRT-PCR might a be more suitable approach than a dedicated array.

Initial Studies on the Occurrence of Cyanobacteria and Microcystins in Irish Lakes

We report the results of a synoptic survey at 14 sites across the north of Ireland undertaken to determine the occurrence of cyanobacteria and their constituent microcystin cyanotoxins. Seven microcystin toxins were tested for, and five of which were found, with MC-LR, MC-RR, and MC-YR being the most prevalent. Gomphosphaeria spp and Microcystis aeruginosa were the most dominant cyanobacterial species encountered. Together with Aphanizomenon flos-aquae, these were the cyanobacteria associated with the highest microcystin concentrations. The occurrence of several microcystin toxins indicates that there may potentially be more than one cyanobacteria species producing microcystins at many sites. Total microcystin concentrations varied over three orders of magnitude dividing the sites into two groups of high (>1000 ngMC/μgChla, six sites) or low toxicity (<200 ngMC/μgChla, eight sites). © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2010.

An in vitro investigation of endocrine disrupting effects of tricothecenes deoxynivalenol (DON), T-2, HT-2 toxins

Trichothecenes are a large family of chemically related mycotoxins. Deoxynivalenol (DON), T-2 and HT-2 toxins belong to this family and are produced by various species of Fusarium. The H295R steroidogenesis assay, regulation of steroidogenic gene expression and reporter gene assays (RGAs) for the detection of androgen, estrogen, progestagen and glucocorticoid (ant)agonist responses, have been used to assess the endocrine disrupting activity of DON, T-2 and HT-2 toxins.

H295R cells were used as a model for steroidogenesis and gene expression studies and exposed with either DON (0.1–1000 ng/ml), T-2 toxin (0.0005–5 ng/ml) or HT-2 toxin (0.005–50 ng/ml) for 48 h. We observed a reduction in hormone levels in media of exposed cells following radioimmunoassay. Cell viability was determined by four colorimetric assays and we observed reduced cell viability with increasing toxin concentrations partly explaining the significant reduction in hormone levels at the highest toxin concentration of all three trichothecenes.

Thirteen of the 16 steroidogenic genes analyzed by quantitative real time PCR (RT-qPCR) were significantly regulated (P < 0.05) by DON (100 ng/ml), T-2 toxin (0.5 ng/ml) and HT-2 toxin (5 ng/ml) compared to the control, with reference genes (B2M, ATP5B and ACTB). Whereas HMGR and CYP19 were down-regulated, CYP1A1 and CYP21 were up-regulated by all three trichothecenes. DON further up-regulated CYP17, HSD3B2, CYP11B2 and CYP11B1 and down-regulated NR5A1. T-2 toxin caused down-regulation of NR0B1 and NR5A1 whereas HT-2 toxin induced up-regulation of EPHX and HSD17B1 and down-regulation of CYP11A and CYP17. The expressions of MC2R, StAR and HSD17B4 genes were not significantly affected by any of the trichothecenes in the present study.

Although the results indicate that there is no evidence to suggest that DON, T-2 and HT-2 toxins directly interact with the steroid hormone receptors to cause endocrine disruption, the present findings indicate that exposure to DON, T-2 toxin and HT-2 toxin have effects on cell viability, steroidogenesis and alteration in gene expression indicating their potential as endocrine disruptors.

Detection of Paralytic Shellfish Toxins by a Solid-Phase Inhibition Immunoassay Using a Microsphere-Flow Cytometry System

Paralytic shellfish poisoning is a toxic syndrome described in humans following the ingestion of seafood contaminated with saxitoxin and/or its derivatives. The presence of these toxins in shellfish is considered an important health threat and their levels in seafood destined to human consumption are regulated in many countries, as well as the levels of other chemically unrelated toxins. We studied the feasibility of immunodetection of saxitoxin and its analogs using a solid-phase microsphere assay coupled to flow cytometry detection in a Luminex 200 system. The technique consists of a competition assay where the toxins in solution compete with bead-bound saxitoxin for binding to an antigonyautoxin 2/3 monoclonal antibody (GT-13A). The assay allowed the detection of saxitoxin both in buffer and mussel extracts in the range of 2.2-19.7 ng/mL (IC(20)-IC(80)). Moreover, the assay cross-reactivity with other toxins of the group is similar to previously published immunoassays, with adequate detection of most analogs except N-1 hydroxy analogs. The recovery rate of the assay for saxitoxin was close to 100%. This microsphere-based immunoassay is suitable to be used as a screening method, detecting saxitoxin from 260 to 2360 µg/kg. This microsphere/flow cytometry system provided similar sensitivities to previously published immunoassays and provides a solid background for the development of easy, flexible multiplexing of toxin detection in one sample.

Development and validation of an ultrasensitive fluorescence planar waveguide biosensor for the detection of paralytic shellfish toxins in marine algae

Marine dinoflagellates of the genera Alexandrium are well known producers of the potent neurotoxic paralytic shellfish toxins that can enter the food web and ultimately present a serious risk to public health in addition to causing huge economic losses. Direct coastal monitoring of Alexandrium spp. can provide early warning of potential shellfish contamination and risks to consumers and so a rapid, sensitive, portable and easy-to-use assay has been developed for this purpose using an innovative planar waveguide device. The disposable planar waveguide is comprised of a transparent substrate onto which an array of toxin-protein conjugates is deposited, assembled in a cartridge allowing the introduction of sample, and detection reagents. The competitive assay format uses a high affinity antibody to paralytic shellfish toxins with a detection signal generated via a fluorescently labelled secondary antibody. The waveguide cartridge is analysed by a simple reader device and results are displayed on a laptop computer. Assay speed has been optimised to enable measurement within 15 min. A rapid, portable sample preparation technique was developed for Alexandrium spp. in seawater to ensure analysis was completed within a short period of time. The assay was validated and the LOD and CCß were determined as 12 pg/mL and 20 pg/mL respectively with an intra-assay CV of 11.3% at the CCß and an average recovery of 106%. The highly innovative assay was proven to accurately detect toxin presence in algae sampled from the US and European waters at an unprecedented cell density of 10 cells/L. © 2012 Elsevier B.V. All rights reserved.

Next generation planar waveguide detection of microcystins in freshwater and cyanobacterial extracts, utilising a novel lysis method for portable sample preparation and analysis

The study details the development of a fully validated, rapid and portable sensor based method for the on-site analysis of microcystins in freshwater samples. The process employs a novel lysis method for the mechanical lysis of cyanobacterial cells, with glass beads and a handheld frother in only 10min. The assay utilises an innovative planar waveguide device that, via an evanescent wave excites fluorescent probes, for amplification of signal in a competitive immunoassay, using an anti-microcystin monoclonal with cross-reactivity against the most common, and toxic variants. Validation of the assay showed the limit of detection (LOD) to be 0.78ngmL and the CCß to be 1ngmL. Robustness of the assay was demonstrated by intra- and inter-assay testing. Intra-assay analysis had % C.V.s between 8 and 26% and recoveries between 73 and 101%, with inter-assay analysis demonstrating % C.V.s between 5 and 14% and recoveries between 78 and 91%. Comparison with LC-MS/MS showed a high correlation (R=0.9954) between the calculated concentrations of 5 different Microcystis aeruginosa cultures for total microcystin content. Total microcystin content was ascertained by the individual measurement of free and cell-bound microcystins. Free microcystins can be measured to 1ngmL, and with a 10-fold concentration step in the intracellular microcystin protocol (which brings the sample within the range of the calibration curve), intracellular pools may be determined to 0.1ngmL. This allows the determination of microcystins at and below the World Health Organisation (WHO) guideline value of 1µgL. This sensor represents a major advancement in portable analysis capabilities and has the potential for numerous other applications.

Multi-detection of Paralytic, Diarrheic and Amnesic Shellfish Toxins by an Inhibition Immunoassay Using a Microsphere-Flow Cytometry System

The presence of paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP) toxins in seafood is a severe and growing threat to human health. In order to minimize the risks of human exposure, the maximum content of these toxins in seafood has been limited by legal regulations worldwide. The regulated limits are established in equivalents of the main representatives of the groups: saxitoxin (STX), okadaic acid (OA) and domoic acid (DA), for PSP, DSP and ASP, respectively. In this study a multi-detection method to screen shellfish samples for the presence of these toxins simultaneously was developed. Multiplexing was achieved using a solid-phase microsphere assay coupled to flow-fluorimetry detection, based on the Luminex xMap technology. The multi-detection method consists of three simultaneous competition immunoassays. Free toxins in solution compete with STX, OA or DA immobilized on the surface of three different classes of microspheres for binding to specific monoclonal antibodies. The IC50 obtained in buffer was similar in single- and multi-detection: 5.6 ± 1.1 ng/mL for STX, 1.1 ± 0.03 ng/mL for OA and 1.9 ± 0.1 ng/mL for DA. The sample preparation protocol was optimized for the simultaneous extraction of STX, OA and DA with a mixture of methanol and acetate buffer. The three immunoassays performed well with mussel and scallop matrixes displaying adequate dynamic ranges and recovery rates (around 90 % for STX, 80 % for OA and 100 % for DA). This microsphere-based multi-detection immunoassay provides an easy and rapid screening method capable of detecting simultaneously in the same sample three regulated groups of marine toxins.

Studies in the use of magnetic microspheres for immunoaffinity extraction of paralytic shellfish poisoning toxins from shellfish

Paralytic shellfish poisoning (PSP) is a potentially fatal human health condition caused by the consumption of shellfish containing high levels of PSP toxins. Toxin extraction from shellfish and from algal cultures for use as standards and analysis by alternative analytical monitoring methods to the mouse bioassay is extensive and laborious. This study investigated whether a selected MAb antibody could be coupled to a novel form of magnetic microsphere (hollow glass magnetic microspheres, brand name Ferrospheres-N) and whether these coated microspheres could be utilized in the extraction of low concentrations of the PSP toxin, STX, from potential extraction buffers and spiked mussel extracts. The feasibility of utilizing a mass of 25 mg of Ferrospheres-N, as a simple extraction procedure for STX from spiked sodium acetate buffer, spiked PBS buffer and spiked mussel extracts was determined. The effects of a range of toxin concentrations (20-300 ng/mL), incubation times and temperature on the capability of the immuno-capture of the STX from the spiked mussel extracts were investigated. Finally, the coated microspheres were tested to determine their efficiency at extracting PSP toxins from naturally contaminated mussel samples. Toxin recovery after each experiment was determined by HPLC analysis. This study on using a highly novel immunoaffinity based extraction procedure, using STX as a model, has indicated that it could be a convenient alternative to conventional extraction procedures used in toxin purification prior to sample analysis.

Production of a broad specificity antibody for the development and validation of an optical SPR screening method for free and intracellular microcystins and nodularin in cyanobacteria cultures

A highly sensitive broad specificity monoclonal antibody was produced and characterised for microcystin detection through the development of a rapid surface plasmon resonance (SPR) optical biosensor based immunoassay. The antibody displayed the following cross-reactivity: MC-LR 100%; MC-RR 108%; MC-YR 68%; MC-LA 69%; MC-LW 71%; MC-LF 68%; and Nodularin 94%. Microcystin-LR was covalently attached to a CM5 chip and with the monoclonal antibody was employed in a competitive 4min injection assay to detect total microcystins in water samples below the WHO recommended limit (1µg/L). A 'total microcystin' level was determined by measuring free and intracellular concentrations in cyanobacterial culture samples as this toxin is an endotoxin. Glass bead beating was used to lyse the cells as a rapid extraction procedure. This method was validated according to European Commission Decision 96/23/EC criteria. The method was proven to measure intracellular microcystin levels, the main source of the toxin, which often goes undetected by other analytical procedures and is advantageous in that it can be used for the monitoring of blooms to provide an early warning of toxicity. It was shown to be repeatable and reproducible, with recoveries from spiked samples ranging from 74 to 123%, and had % CVs below 10% for intra-assay analysis and 15% for inter-assay analysis. The detection capability of the assay was calculated as 0.5ng/mL for extracellular toxins and 0.05ng/mL for intracellular microcystins. A comparison of the SPR method with LC-MS/MS was achieved by testing six Microcystis aeruginosa cultures and this study yielded a correlation R(2) value of 0.9989.

The Application of Semiconductor Photocatalysis for the Removal of Cyanotoxins from Water and Design Concepts for Solar Photocatalytic Reactors for Large Scale Water Treatment

There has been a significant increase in the occurrence of cyanobacterial blooms in freshwaters over the past few decades due to escalating nutrient levels. These cyanobacteria release a range of toxins, for example microcystins which are chemically very stable. Many cyanotoxins are consequently very difficult to remove from water using existing treatment technologies. Semiconductor photocatalysis, however, has proven to be a very effective process for the removal of these compounds from water. In this chapter we consider the application of this highly versatile and exciting technology for the decomposition of cyanotoxins. Furthermore design concepts for solar photocatalytic reactors that could be utilized for the removal of these toxins are also considered