Detection Of Toxins Research Articles

MXene-based aptasensor for the detection of aflatoxin in food and agricultural products

The detection of toxins that contaminate food needs highly sensitive and selective techniques to prevent substantial monitory loss. In this regard, various nanostructured material-enabled biosensors, have recently been developed to improve the detection of food toxins among them aflatoxin is the prevalent one. The biosensor-based detection of aflatoxin is quick, cheaper, and needs less skilled personnel, therefore overcoming the shortcomings of conventional techniques such as LC/MS-MS, HPLC, and ELISA assays. 2D MXenes manifest as an efficient material for biosensing due to their desirable biocompatibility, magnificent mechanical strength, easiness of surface functionalization, and tuneable optical and electronic features. Contrary to this, aptamers as biorecognition elements (BREs) possess high selectivity, sensitivity, and ease of synthesis when compared to conventional BREs. In this review, we explored the most cutting-edge aptamer-based MXene-enabled biosensing technologies for the detection of the most poisonous mycotoxins (i.e., Aflatoxins) in food and environmental matrices. The discussion begins with the synthesis processes and surface functionalization/modification of MXenes. Computational approaches for designing aptasensors and advanced data analysis based on artificial intelligence and machine learning with special emphasis over Internet-of-Thing integrated biosensing devices has been presented. Besides, the advantages of aptasensors over conventional methods along with their limitations have been briefed. Their benefits, drawbacks, and future potential are discussed concerning their analytical performance, utility, and on-site adaptability. Additionally, next-generation MXene-enabled biosensing technologies that provide end users with simple handling and improved sensitivity and selectivity have been emphasized. Owing to massive applicability, economic/commercial potential of MXene in current and future perspective have been highlighted. Finally, the existing difficulties are scrutinized and a roadmap for developing sophisticated biosensing technologies to detect toxins in various samples in the future is projected.

Identification and Characterization of Clostridium perfringens Atypical CPB2 Toxin in Cell Cultures and Field Samples Using Monoclonal Antibodies.

A direct sandwich enzyme-linked immunosorbent assay (sELISA) was developed for the detection of the atypical β2-toxin (CPB2) of Clostridium perfringens. Polyclonal (PAbs) and monoclonal (MAbs) antibodies were previously obtained employing recombinant CPB2 produced in the baculovirus system as antigen. In the current study, PAbs were used as capture molecules, while purified MAbs conjugated to horseradish peroxidase (MAbs-HRP) were used for the detection of atypical CPB2 toxin. MAbs 5C11E6 and 2G3G6 showed high reactivity, sensitivity and specificity when tested on 232 C. perfringens cell culture isolates. In addition, a reactivity variation among different strains producing atypical CPB2 toxin was observed using the conformation-dependent MAb 23E6E6, suggesting the hypothesis of high instability and/or the existence of different three-dimensional structures of this toxin. Results obtained by sELISA and Western blotting performed on experimentally CPB2-contaminated feces revealed a time-dependent proteolytic degradation as previously observed with the consensus allelic form of CPB2. Finally, the sELISA and an end-point PCR, specific for the atypical cpb2 gene, were used to test field samples (feces, rectal swabs and intestinal contents) from different dead animal species with suspected or confirmed clostridiosis. The comparison of sELISA data with those obtained with end-point PCR suggests this method as a promising tool for the detection of atypical CPB2 toxin.

Comparative Evaluation of Luminex xTAG® Gastrointestinal Pathogen Panel and Direct-From-Stool Real-Time PCR for Detection of C. difficile Toxin tcdB in Stool Samples from a Pediatric Population.

Detection of Clostridioides difficile toxins in patients with gastroenteritis has increasingly been accomplished through the use of enteric multiplex syndromic panels. Comparisons of the performance of these panels to both direct-from-stool (DFS) and culture-enriched stools followed by polymerase chain reaction (PCR) methods in pediatric populations are limited. Here, we compare the performance of the Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) to our DFS in-house real-time PCR (DFS RT-PCR) assay for the detection of C. difficile toxin gene, tcdB, using 2641 stool specimens collected from children enrolled in the Alberta Provincial Pediatric EnTeric Infection Team (APPETITE) study in Alberta, Canada. We used culture enrichment followed by in-house RT-PCR to resolve discordant results between the two assays. We found excellent agreement (k = 0.89) between the GPP and our DFS RT-PCR assay: the positive percent agreement between the two assays was 97%, and the negative percent agreement was 99%. GPP, a multi-analyte platform can easily be implemented into a routine diagnostic laboratory for detecting enteric pathogens including C. difficile.

Five new species of Inosperma from China: Morphological characteristics, phylogenetic analyses, and toxin detection.

Many species of Inosperma cause neurotoxic poisoning in humans after consumption around the world. However, the toxic species of Inosperma and its toxin content remain unclear. In the present study, we proposed five new Inosperma species from China, namely, I. longisporum, I. nivalellum, I. sphaerobulbosum, I. squamulosobrunneum, and I. squamulosohinnuleum. Morphological and molecular phylogenetic analyses based on three genes (ITS, nrLSU, rpb2) revealed that these taxa are independent species. A key to 17 species of Inosperma in China is provided. In addition, targeted screening for the most notorious mushroom neurotoxins, muscarine, psilocybin, ibotenic acid, and muscimol, in these five new species was performed by using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Our results show that the neurotoxin contents in these five species varied: I. sphaerobulbosum contains none of the tested neurotoxins; I. nivalellum is muscarine positive; I. longisporum and I. squamulosohinnuleum contain both ibotenic acid and muscimol, and I. squamulosobrunneum only contains muscimol; psilocybin was not detected in these five new species.

High-performance and -efficiency cardiomyocyte-based potential biosensor for temporal-specific detection of ion channel marine toxins

Paralytic shellfish toxins (e.g., saxitoxin, STX; gonyautoxin-2, GTX-2) and tetrodotoxin (TTX) are highly toxic and widely distributed ion channel marine toxins which specifically block the voltage-dependent sodium channels (VDSCs), causing great harm to human health. It is urgent to exploit new detection methods with high specificity and high efficiency. Here, a portable high-throughput cardiomyocyte-based potential biosensor was established with cardiomyocytes, a 16-well microelectrodes (MEs) sensor and a robust 32-channel recording system, which presented high-quality and high-consistency extracellular field potential (EFP) signals in each well with a long duration of 80 h. The feature parameters, including firing rate (FR), spike amplitude (SA), spike slope (SS), spike duration (SD) and field potential duration (FPD), were extracted from EFP to quantitatively assess the toxic effects of these ion channel toxins. Importantly, the biosensor showed temporal specificity and parametric selectivity under toxin treatments, and FR, SS and SD were the optimal parameters to STX, TTX and GTX-2, respectively. This biosensor can rapidly detect 0.29 ng/mL STX, 0.30 ng/mL TTX and 0.16 ng/mL GTX-2 within 5 min, 10 min and 15 min, respectively. Thus, our novel multi-well cardiomyocyte-based biosensor will be a promising tool for high-effective detection of ion channel toxins.

Phage-displayed nanobody-based fluorescence-linked immunosorbent assay for the detection of Cry3Bb toxin in corn

Cry toxins have been widely used as biopesticides and applied in transgenic crops for the control of pests, raising public concerns for unforeseen environmental and health risks. In this study, five phage-displayed nanobodies against Cry3Bb toxin were selected from a naive nanobody library by screening. Using purified phage-displayed nanobody as capture antibody, a novel double-antibody sandwich fluorescence-linked immunosorbent assay (FLISA) based on quantum dot beads (QBs) was established for the determination of Cry3Bb toxin. The developed method exhibited a linear working range of 31.25–500 ng/mL and a low detection limit of 8.45 ng/mL. The recoveries of Cry3Bb toxin from spiked corn samples were ranging from 93.1% to 116.8% with a coefficient of variation lower than 9.4%. Molecular modeling results showed that key amino acids in the CDR3 region of nanobody P27 combine with Cry3Bb toxin through hydrogen bonds and hydrophobic interactions. These results indicated that the phage-displayed nanobody-based QBs-FLISA may provide a possible tool for detecting Cry3Bb toxin in corn.

Modular, Portable, Automatically, Rapidly, Sensitive, and Selectively Essence Sensor System

The ESSENCE platform is an electrochemical sensor system that utilizes non-planar porous microelectrode microfluidic chips with a fully programmable fluidic/sample system to be a “portable, automatic, rapid, sensitive, and selective” for different emerging contaminants and liquid biopsy. The system is designed with non-planar porous microelectrode interfaces, containing different functional capturing probes for specific sensing tasks. From our previous report, this 3D non-planar structure has advantages such as increased flow’s convective fluxes and disrupts diffusive processes like the electric double layer (EDL). The porous electrode structure utilized the packing material’s higher Zeta potential than the bare glass surface, resulting in the EDL length will be significantly smaller than the clear channel. Due to a smaller EDL length, the relaxation frequency will increase, which shifts the EIS signal to a higher frequency range (1kHz to 100MHz) and results in a fast response with a higher signal-to-noise ratio in Electrochemical Impedance Spectroscopy (EIS) measurements. From the perspective of electric field penetration, the top-down electric direction covers all the packing sensing areas, providing a generally higher electronic signal response than the traditional planar electrode system, leading to a higher signal-to-noise ratio. A second feature from the perspective of microfluidics is the significant fluidic shear force generated due to flow through the porous electrode surface. This higher shear force on the surface can unbind most false-positive signals generated by non-specific binding, such as physical adhesive or biofouling. These unique benefits provide physical enhancements of sensitivity and selectivity without manipulating the chemical material in the system. Earlier we showed that our detection of the biomolecules like DNA (fM sensitivity, which can distinguish against a mismatched DNA), proteins (breast cancer biomarker p53, fM sensitivity, which can distinguish it against another breast cancer biomarker HER2), PFOS (Perfluorooctanesulfonate, 0.5 ng) under the traditional desktop Electrochemical Impedance Spectroscopy (EIS) analyzer in the total process of hourly. In this talk, we have further developed our previous ESSENCE platform to a modular, automatic, and portable, one-stop instrument for clinicians to screen for infectious diseases, liquid biopsy, and toxin detection to detect emerging pathogens while significantly reducing false positives and false negatives, but also decreasing our detecting process to 15 mins in much more hazard background conditions to meet the criteria of POC. Second, A small (15 cm x 17cm) USB programable fluidic/oscilloscope base with a 15 minutes total detecting protocol is applied to our ESSENCE system for a fast Point-of-care (POC) diagnostic. This portable ESSENCE platform can respond to the measurable EIS signals from the oscilloscope with 25 ul samples size (DNA and HER2 proteins in undiluted artificial Urine) with a fully automatic process at the uM to pM regions in 15 minutes. Each ESSENCE chip is also significantly cheaper than the ELISA; the chip costs about $20 (for an academic lab) and can be reused. Figure 1

Multifunctional Smart ZnSe-Nanostructure-Based Fluorescent Aptasensor for the Detection of Ochratoxin A.

Herein, we present a comprehensive investigation of rationally designed zinc selenide (ZnSe) nanostructures to achieve highly negatively charged ZnSe nanostructures. A Microwave-assisted hydrothermal synthesis method was used to synthesize three types of ZnSe nanostructures, i.e., nanorods, µ-spheres and nanoclusters, as characterized by a zeta potential analyzer, X-ray diffraction (XRD), scanning electron microscopy (SEM), Raman spectroscopy and BET, which were labeled as type A, B and C. Three different solvents were used for the synthesis of type A, B and C ZnSe nanostructures, keeping other synthesis conditions such as temperature, pressure and precursors ratio constant. Based on two heating time intervals, 6 and 9 h, types A, B and C were further divided into types A6, A9, B6, B9, C6 and C9. ZnSe nanostructures were further evaluated based on their fluorescent quenching efficiency. The maximum fluorescence quenching effect was exhibited by the ZnSe-B6 type, which can be attributed to its highly negative surface charge that favored its strong interaction with cationic dye Rhodamine B (Rh-B). Further, the optimized ZnSe-B6 was used to fabricate an aptasensor for the detection of a food-based toxin, ochratoxin-A (OTA). The developed aptasensor exhibited a limit of detection of 0.07 ng/L with a wide linear range of 0.1 to 200 ng/L.

Electrochemical sensing platform for the detection of nitroaromatics using g-C3N4/V2O5 nanocomposites modified glassy carbon electrode

Rapid, reliable, cost-effective and trace-level detection of nitroaromatic toxins is becoming increasingly important because of their carcinogenic nature, and their presence in water sources as an environmental contaminant. The present study reports the development of an electrochemical sensing platform based on g-C3N4/V2O5 (VOCN) nanocomposites modified glassy carbon electrode (GCE) for the effective detection of nitroaromatics. The VOCN nanocomposites were synthesized via wet impregnation method, and were characterized by XRD, FT-IR, HRSEM and XPS techniques. The electrochemical behavior of four nitroaromatics (4-NP, DNP, DNT and TNP) were investigated on seven nanomaterials-based sensors using cyclic voltammetry, square wave voltammetry and chronoamperometry techniques. Under optimized conditions, the nitroaromatic compounds exhibited reduction peaks corresponding to the number of nitro-functional groups on an aromatic ring. The electrochemical studies revealed that the composite with 7 wt% V2O5 (VOCN7/GCE) exhibits higher sensitivity for the detection of 4-NP, DNP, DNT and TNP. The VOCN7/GCE recorded the limit of detection (LOD) as low as 0.85 nM, 3.3 nM, 3.4 nM and 11.0 nM in a linear range between 1.0 nM - 100.0 µM, 10.0 nM - 100.0 µM, 10.0 nM - 100.0 µM and 30.0 nM - 100.0 µM for 4-NP, DNP, DNT and TNP, respectively. The fabricated sensor showed high reusability and reproducibility, and could detect the four nitroaromatics in real water samples.

FeMOF-based nanostructured platforms for T-2 toxin detection in beer by a "fence-type" aptasensing principle.

In this work, a label-free electrochemical aptasensor for the detection of T-2 toxin was constructed, using gold nanoparticles/Fe-based metal organic framework@graphene oxide (AuNPs/FeMOF@GO) nanocomposites, exonuclease (RecJf), and the "fence-type" structure of aptamer-single stranded DNA (Apt-sDNA) complex as the signal amplification element. Wherein, AuNPs/FeMOF@GO nanocomposite effectively improves the aptasensor performance by improving the electron transfer capacity of the electrode and providing a larger specific surface area to load a large number of Apt-sDNA structures. Meanwhile, the shear effect of RecJf, which was induced by T-2 toxin, further improves the analytical performance of the aptasensor. Under the optimum conditions, the constructed aptasensor shown excellent performance for T-2 toxin detection, with a wide linear range (5.0 × 10-1pg·mL-1-5.0 × 106pg·mL-1) and a low limit of detection of 0.19pgmL-1. Also, the aptasensor showed high specificity, excellent stability, and available repeatability. What's more, the prepared aptasensor was explored for its application in actual samples and showed good detection results, which provided a new strategy for detecting T-2 toxin in the food and feed. A label-free electrochemical aptasensor for the detection of T-2 toxin was constructed using gold nanoparticles/Fe-metal organic framework@graphene oxide (AuNPs/FeMOF@GO) nanocomposites, exonuclease (RecJf), and the "fence-type" structure of aptamer-single stranded DNA (Apt-sDNA) complex as the signal amplification element.

Triple immunochromatographic test system for detection of priority aquatic toxins in water and fish.

Aquatic toxins are a group of toxic compounds produced by several types of freshwater and marine algae and cyanobacteria and transported through the food chains of water bodies. Potential contamination of aquaculture products (raw and processed fish and seafood) with aquatic toxins requires the use of efficient screening methods for their control. In this study, a multiplex immunochromatographic test system for the simultaneous detection of three aquatic toxins-phycotoxins domoic acid (DA) and okadaic acid (OA), and cyanotoxin microcystin-LR (MC-LR)-is for the first time developed. For this, a competitive indirect immunochromatographic analysis (ICA) based on gold-labeled secondary antibodies was carried out. The LODs/cutoffs/working ranges of the ICA were 0.05/0.3/0.07-0.29, 1.3/100/3.2-58.2, and 0.1/2.0/0.2-1.1ng/mL for MC-LR, DA, and OA, respectively. The assay duration was 18min. The developed test system was used to analyze water samples from natural sources (salt and fresh water) and fish samples. For sample preparation of water, simple dilution with a buffer was proposed; for fish samples, methanol-water extraction was utilized. It was demonstrated that the triple LFIA specifically detected target aquatic toxins with recoveries of 85.0-121.5%. The developed multiplex LFIA can be considered a promising analytical solution for the rapid, easy, and sensitive control of water and food safety.

Ultra-sensitive electrochemiluminescence biosensor for abrin detection based on dual-labeled phage display affibodies and polystyrene nanospheres

Abrin is a plant cytotoxin that poses a high threat to public safety and therefore requires highly sensitive and efficient detection methods. Affibodymolecules are artificial affinity proteins that have been widely studied in bioimaging and medical treatment but rarely used in bioassays. In this work, an electrochemiluminescence (ECL) biosensor for abrin was established with biotin and Ru(bpy)32+ dual-labeled phage display affibodies as molecular recognition and signal element. On this basis, dual-labeled polystyrene nanospheres (nano-PSs) were chosen to link with phages and further achieve ultra-sensitive detection. This assay has a triple signal amplification. First, capsid proteins of M13 phages have connected numerous signal molecules. Second, the nano-PSs bound to the surface of the phages also contained quite a few signal molecules. Third, with the theoretically infinitely repeatable “biotin - streptavidin (SA) - biotin” composite unit as the “mortise and tenon structure”, the dual-labeled nano-PSs further formed highly stacked nano-aggregates, ultimately achieving significant signal amplification. Besides, the detection platform of this assay is a portable electrochemiluminescence sensor based on the screen-printed electrode, which is small and easy to operate. Limit of detection (LOD) for abrin of the ECL biosensor was 9.3 fg/mL and the linear detection range was observed from 10 fg/mL to 100 pg/mL. This new biosensing system has also demonstrated good reproducibility, specificity, practicability and has great potential for highly sensitive detection of contaminants, toxins and biomarkers in environmental monitoring and clinical application.

A comprehensive review on electrochemical and optical aptasensors for organophosphorus pesticides.

There has been a rise in pesticide use as a result of the growing industrialization of agriculture. Organophosphorus pesticides have been widely applied as agricultural and domestic pest control agents for nearly five decades, and they remain as health and environmental hazards in water supplies, vegetables, fruits, and processed foods causing serious foodborne illness. Thus, the rapid and reliable detection of these harmful organophosphorus toxins with excellent sensitivity and selectivity is of utmost importance. Aptasensors are biosensors based on aptamers, which exhibit exceptional recognition capability for a variety of targets. Aptasensors offer numerous advantages over conventional approaches, including increased sensitivity, selectivity, design flexibility, and cost-effectiveness. As a result, interest in developing aptasensors continues to expand. This paper discusses the historical and modern advancements of aptasensors through the use of nanotechnology to enhance the signal, resulting in high sensitivity and detection accuracy. More importantly, this review summarizes the principles and strategies underlying different organophosphorus aptasensors, including electrochemical, electrochemiluminescent, fluorescent, and colorimetric ones.

Clostridioides difficile toxin is infrequently detected in inflammatory bowel disease and does not associate with clinical outcomes

IntroductionClostridioides difficile infection (CDI) rates and outcomes can vary based on differences in testing strategy. Our aim was to assess the prevalence of toxin detection in inflammatory bowel disease (IBD) when compared to those without IBD. Secondly, the clinical outcomes of CDI in IBD were assessed using two-step testing strategies.MethodsWe included patients undergoing CDI testing from four academic centers in the United States between January 1, 2018 and June 30, 2020. First the prevalence of toxin detection was compared between individuals with IBD and those without IBD. Secondly, among patients with IBD a primary composite outcome of abdominal colectomy, admission to an intensive care unit (ICU) or death within 30 days of C. difficile testing was assessed across the three categorical groups (screen positive/toxin positive, screen positive/toxin negative and screen negative assay) resulting from the two-step testing strategy.ResultsWhen comparing individuals with a positive screening assay, patients with IBD were less likely to have toxin detected by enzyme immunoassay (EIA) as compared to the non-IBD population (22/145 (15.2%) vs. 413/1144 (36.1%), p < 0.0001). Among all patients with IBD (n = 300), twenty-five (8.3%) had a screen positive assay/toxin positive assay, 136 (45.3%) had a screen positive/toxin negative assay and 139 (46.3%) had a negative screening assay. No significant difference in the primary composite outcome was detected across the three groups (p = 0.566).ConclusionWhen compared to those without IBD, patients with IBD have a reduced proportion of cases of C. difficile with toxin positivity. Differences in clinical outcomes among patients with IBD were not detected and limited by the infrequent detection of expressed toxin.

Sensitive time-resolved fluoroimmunoassay for the quantitative detection of okadaic acid

Okadaic acid (OA) is a biotoxin from marine microalgae and widely present in shellfish, which severely affects the seafood safety. Therefore, it is essential to establish a highly sensitive OA analysis and detection method. In this study, a new type of immunoassay technology was established on the basis of the competition method using time-resolved fluoroimmunoassay (TRFIA). OA-bovine serum albumin (OA-BSA) coated on a 96-well plate competes with OA standard or samples to bind OA antibodies. A rare-earth ion-labeled secondary antibody, which fluoresces strongly under the effect of the enhancement solution, was then added as a tracer for detection. The established linear range of OA detected by TRFIA was 2.49 × 10-3 – 50 ng/ml, and the limit of detection was 2.49 × 10-3 ng/ml. The average coefficients of variation from intra-assay and inter-assay batches were 3.34% and 5.87%, respectively, and the recovery rate was 93.04%–111.66%. The OA in shellfish samples was determined by TRFIA and high-performance liquid chromatography (HPLC), and the results showed a good correlation. This study established a TRFIA to detect OA, which has the characteristics of simplicity, sensitivity, precision, and high accuracy, far exceeding the EU or the US standards for the detection of shellfish toxins. It is expected to make proper contribution in marine biotoxin detection.

Designing of immunodiagnostic assay using polyclonal antibodies for detection of Shiga toxin producing pathogenic E. coli (STEC) strains.

Shiga toxin-producingEscherichia coli(STEC) are the leading causes of diarrhea and death of humans worldwide. Many diagnostic assays have been developed to aid for the diagnosis of STEC strain; however, they have limitations. Thus, this study was aimed at designing rapid, effective, sensitive and specific immunodiagnostic assay for STEC strain detection. Thus, a STEC isolate from Ethiopia was processed for LPS extraction and the LPS was used to immunize mice.. The produced antibody showed positive agglutination both on the purified LPS as well as the STEC isolate carrying LPS on their surface; however, agglutination of STEC was more pronounced. Mice immunized with LPS produced highest agglutination on tertiary immunization showing the progressive buildup of the antibody response against the antigen. Cultures from tryptone soya agar and when they refresh showed better agglutination than cultures on EMB as well as tryptone soya broth. Immunodiagnostic assay developed in this study could detect STEC strains including STEC in human feces rapidly (1-2min), with high sensitivity (90.2%), specificity (89.5%) and accuracy (90.6%). However, further studies are still required to improve the sensitivity, specificity and reproducibility. Overall this diagnostic assay provided promising results that may curb current problem with detection methods in clinical health care and research laboratories.

Analytical strategy to document horse fatalities related to Oenanthe crocata poisoning

To report the analytical procedure that was developed and applied to identify oenanthotoxin (OT) and its metabolite 2,3-dihydro-oenanthotoxin (2,3DHOT) in biological and vegetal samples using both LC-DAD and LC-HRMS methods in two cases of equine fatal intoxication by Oenanthe crocata. : (case#1) a 7-year-old Arabian horse was found dead within one hour after the ingestion of «wild carrots», and was autopsied the following day. The gross and microscopic examination was unremarkable, except the presence of several root pieces in the animal's stomach. Sections of the same roots found on the site showed hexagonal subdivisions and a yellowish latex. These findings were consistent with Hemlock water-dropwort (Oenanthe crocata) and confirmed by an experienced botanist. (case#2) an 8-year-old pony was unexpectedly found dead and autopsied two days later. The body conservation was poor; the only significant finding was the presence of several root pieces of undetermined origin in the stomach. Autopsy samples (root pieces found in the stomach, stomach content, liver, kidney, and spleen of case#1; stomach content of case#2) were stored at -20 °C before analysis, while the on-site roots (case#1) were stored at room temperature. After grinding organ samples, 1 to 10 g were extracted using methanol (5 mL), vortex mixed for 30 seconds, and kept at 4 °C for 2 hours. These methanolic suspensions were centrifuged firstly at 2500 g for 10 min, then the supernatant was secondly centrifugated at 8000 g for 14 min. In order to enhance detection sensitivity, one mL of each supernatant from the liver, kidney, and spleen samples was dried under nitrogen and then each extract was resuspended with 100 μL of methanol. Extracts were analysed using two analytical methods (LC-DAD and LC-HRMS) both using the same ACQUITY HSS C18 column (150 × 2 mm, 20 μm) for chromatographic separation. The acquired data were analysed using Empower 3 (LC-DAD) and Unify and Progenesis QI softwares (LC-HRMS) for investigation of specific fragmentation spectra of identified substances. Thanks to combined analytical data (i.e. characteristic UV spectra [Kite. J Chrom B. 2006;838:63-70.] together with specific fragmentation HRMS spectra), oenanthotoxin (OT) and its hydrogenated metabolite, 2,3-dihydro-oenanthotoxin (2,3DHOT*), were identified (case#1) in roots found on the siteand in the stomach content, in the stomach content, kidney, and liver (*also in spleen), and (case#2) in the stomach content. In France, three species of the Apiaceae family can cause life threatening poisoning: the Hemlock (Cicuta virosa), the big Hemlock (Conium maculatum) and the Hemlock water-dropwort (Oenanthe crocata). In the latter one, OT and 2,3DHOT act as non-competitive antagonists of GABA receptors. Clinical signs of poisoning in horses (non-vomiting animals) are dominated by hypersalivation, mydriasis, muscle tremors, respiratory distress, and convulsions, but sometimes sudden death is observed. In humans, the clinical signs are completed/supplemented by ataxia, bruxism, hallucinations, nausea, and vomiting seizures; they occur between 15 min and 10 hours after ingestion [Schep. Clin Tox.2009;47:270–278.]. Analytical detection of Oenanthe crocata toxins in biological samples is a challenge due to their instability (e.g. OT and 2,3DHOT stabilities in post-mortem samples is unknown), the lack of commercially available analytical standards, and the exceptionally low response of these toxins using mass spectrometry detection. Nevertheless, identification of OT and 2,3DHOT can be performed using a combination of LC-DAD and LC-HMRS (for enhanced detection in organs, e.g. liver) methods, in order to support poisoning diagnosis. In this context, we consider that the reported analytical data can be helpful to diagnose oenanthotoxin poisonings.

Detection of toxins and harmful algal bloom cells in shellfish hatcheries and efforts toward removal

As the start of the supply chain for the aquaculture industry, hatcheries are a crucial component in the success of oyster and northern quahog (hard clam) aquaculture on the East Coast of the US. Intermittent failures in hatchery production slow industry growth and reduce profits. To begin investigations into the possible role of algal toxins in hatchery production failure, post-treatment hatchery water from one research and four commercial hatcheries in lower Chesapeake Bay, USA, was sampled for (1) toxin presence and (2) harmful algal bloom (HAB) cell enumeration. Overall, seven toxin classes, likely produced by six different HAB species, were detected in post-treatment hatchery water, despite a lack of visually identifiable HAB cells within the facility. Toxins detected include pectenotoxin-2, goniodomin A, karlotoxin-1 and karlotoxin-3, okadaic acid and dinophysistoxin-1, azaspiracid-1 and azaspiracid-2, brevetoxin-2, and microcystin-LR. In a second, more targeted study, two batches of source water were followed and sampled at each step of a water-treatment process in the VIMS Aquaculture Genetics and Breeding Technology Center research hatchery in Gloucester Point, Virginia, USA. Two treatment steps showed particular promise for decreasing the concentrations of the three toxins detected in the source water, 24-h circulation through sand filters and activated charcoal filtration. Toxin concentrations of pectenotoxin-2, 3.53 ± 0.56 pg mL−1, okadaic acid, 6.14 ± 0.69 pg mL−1, and dinophysistoxin-1, 1.88 ± 0.0 pg mL−1, were low in the source water. The sand filtration step decreased these concentrations by 49–62%. Activated charcoal filtration subsequently brought the concentrations down to <0.5 pg mL−1, successfully removing another 87–99% of toxins from incoming water. With toxin breakthrough now documented in commercial hatchery facilities during non-bloom conditions, future studies are needed to investigate breakthrough and water-treatment options during more-intense bloom conditions, as well as the potential interactions of algal toxins with other stressors in a potentially multifactorial etiology underlying hatchery production failures.

Multiplex Lateral Flow Assay and the Sample Preparation Method for the Simultaneous Detection of Three Marine Toxins.

A multiplex lateral flow immunoassay (LFA) has been developed to detect the primary marine biotoxin groups: amnesic shellfish poisoning toxins, paralytic shellfish poisoning toxins, and diarrhetic shellfish poisoning toxins. The performance characteristics of the multiplex LFA were evaluated for its suitability as a screening method for the detection of toxins in shellfish. The marine toxin-specific antibodies were class-specific, and there was no cross-reactivity between the three toxin groups. The test is capable of detecting all three marine toxin groups, with working ranges of 0.2–1.5, 2.5–65.0, and 8.2–140.3 ng/mL for okadaic acid, saxitoxin, and domoic acid, respectively. This allows the multiplex LFA to detect all three toxin groups at the EU regulatory limits, with a single sample extraction method and dilution volume. No matrix effects were observed on the performance of the LFA with mussel samples spiked with toxins. The developed LFA uses a simple and pocket-sized, portable Cube Reader to provide an accurate result. We also evaluated the use of this Cube Reader with commercially available monoplex lateral flow assays for marine toxins.

Electrochemical aptasensor based on the target-induced strand displacement strategy-driven for T-2 toxin detection

Herein, an aptasensor based on target-induced strand displacement (TISD) strategy was developed for sensitive detection of T-2 toxin. Gold nanoparticles@ aminated manganese dioxide (AuNPs@NH2-MnO2) exhibited excellent electrical conductivity and provided more binding sites for aptamer (Apt). Besides, polyethyleneimine-reduced graphene oxide/gold‑platinum core-shell nanorods composites (PEI-rGO/Pt@Au NRs) were used to be carriers for signaling tags, as their sufficiently large specific surface area improved the loading capacity for signal molecules. In the presence of T-2, the Apt sequence was more inclined to form an Apt-T-2 complex, and the cDNA was displaced from the Apt-cDNA duplex, while the signal tag was released, resulting in a weakened MB signal, differential pulse voltammetry (DPV) was used to record the signal change. Under optimal conditions, the signal response of the constructed electrochemical aptasensor exhibited a good linear relationship with the concentration of T-2. The detection limit was 8.74 × 10−7 ng mL−1over a wide range of concentration from 5 × 10−6 ng mL−1 to 5 ng mL−1. Furthermore, the proposed aptasensor had excellent specificity, good stability and can be well applied to the detection of real samples. It provided a new avenue for the research and development of sensitive aptasensors in food detection and analysis.