Multi-detection method for five common microalgal toxins based on the use of microspheres coupled to a flow-cytometry system

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Analytica Chimica Acta xxx (2014) xxx–xxx

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Multi-detection method for five common microalgal toxins based on the use of microspheres coupled to a flow-cytometry system María Fraga a , Natalia Vilariño a, *, M. Carmen Louzao a , Laura P. Rodríguez a , Amparo Alfonso a , Katrina Campbell b , Christopher T. Elliott b , Palmer Taylor c, Vítor Ramos d, Vítor Vasconcelos d , Luis M. Botana a, * a

Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain Institute for Global Food Security (IGFS), School of Biological Sciences, Queen's University Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG, Northern Ireland, UK c Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA 92093-0657, United States d Interdisciplinary Centre of Marine and Environmental Research, CIIMAR, and Faculty of Sciences, University of Porto, Rua dos Bragas 289, Porto 4050-123, Portugal b

H I G H L I G H T S

G R A P H I C A L A B S T R A C T


Multiplexed method for the detection of five microalgal toxin classes.
Sensitive, easy-to-perform, rapid, semi-quantitative screening technique.
Useful for the detection of freshwater toxins in cyanobacterial samples.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 17 June 2014 Received in revised form 12 August 2014 Accepted 17 August 2014 Available online xxx

Freshwater and brackish microalgal toxins, such as microcystins, cylindrospermopsins, paralytic toxins, anatoxins or other neurotoxins are produced during the overgrowth of certain phytoplankton and benthic cyanobacteria, which includes either prokaryotic or eukaryotic microalgae. Although, further studies are necessary to define the biological role of these toxins, at least some of them are known to be poisonous to humans and wildlife due to their occurrence in these aquatic systems. The World Health Organization (WHO) has established as provisional recommended limit 1 mg of microcystin-LR per liter of drinking water. In this work we present a microsphere-based multi-detection method for five classes of freshwater and brackish toxins: microcystin-LR (MC-LR), cylindrospermopsin (CYN), anatoxin-a (ANA-a), saxitoxin (STX) and domoic acid (DA). Five inhibition assays were developed using different binding proteins and microsphere classes coupled to a flow-cytometry Luminex system. Then, assays were combined in one method for the simultaneousdetectionofthetoxins.TheIC50'susingthismethodwere1.9  0.1 mg L1 MC-LR,1.3  0.1 mg L1 CYN, 61  4 mg L1 ANA-a, 5.4  0.4 mg L1 STX and 4.9  0.9 mg L1 DA. Lyophilized cyanobacterial culture samples were extracted using a simple procedure and analyzed by the Luminex method and by UPLC–IT-TOFMS. Similar quantification was obtained by both methods for all toxins except for ANA-a, whereby the estimated content was lower when using UPLC–IT-TOF-MS. Therefore, this newly developed multiplexed detection method provides a rapid, simple, semi-quantitative screening tool for the simultaneous detection of five environmentally important freshwater and brackish toxins, in buffer and cyanobacterial extracts. ã 2014 Published by Elsevier B.V.

Keywords: Multi-detection Aquatic toxins Microalgal toxins Microsphere-based array Flow-cytometry system Screening method

* Corresponding authors. Tel.: +34 982822233; fax: +34 982822233. E-mail addresses: [email protected] (N. Vilariño), [email protected] (L.M. Botana). http://dx.doi.org/10.1016/j.aca.2014.08.030 0003-2670/ ã 2014 Published by Elsevier B.V.

Please cite this article in press as: M. Fraga, et al., Multi-detection method for five common microalgal toxins based on the use of microspheres coupled to a flow-cytometry system, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.08.030

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1. Introduction Many phytoplankton species belonging to different groups of microalgae, such as diatoms, dinoflagellates or cyanobacteria (formerly known as blue–green algae) have been described as toxin producers in freshwater, brackish and marine ecosystems [1,2]. Cyanobacteria have a wide geographical distribution and include numerous genera involved in the production of toxins (i.e., cyanotoxins), mainly in freshwater environments [3]. Cyanobacterial harmful algal blooms have been associated with human and animal poisonings, which highlights the hazard to human health, the environment and the economy associated to freshwater resources. Cyanotoxins include microcystins (MCs), cylindrospermopsin (CYN), anatoxin-a (ANA-a) and saxitoxin (STX), among few others [4]. Additionally, the marine toxin domoic acid (DA) has been also described in brackish waters. In this case, the microalgal producers are diatoms that belong to the genera Pseudo-nitzschia and Nitzschia [2]. These toxins are usually classified by their chemical structures (Fig. 1) and toxin activity. MCs are cyclic heptapeptides that share a general D-Ala-X-D-MeAsp-Z-Adda-D-Glu-Mdha structure [5]. Variations of the peptides yield about 90 structural variants of MCs [6]. The amino acids Adda, D-Glu and Mdha play an important role in the interaction with the catalytic center of protein phosphatases and therefore, these toxins block the access of the enzyme substrate [7]. CYN is a tricyclic alkaloid with a natural epimer, 7-epicylindrospermopsin, and one analog occurring at the C7 hydroxyl group, 7-deoxycylindrospermopsin (deoxyCYN) [8,9]. CYN has been described as a protein synthesis inhibitor that affects mainly the liver [10]. ANA-a is a potent agonist of the nicotinic acetylcholine receptors (nAChR) [11,12]. ANA-a and its analog, called homoanatoxin-a, are unstable so they will be oxidized and converted into epoxy and dihydro degradation products, depending on the environmental conditions [13,14]. Paralytic shellfish poisoning toxins (PSTs) are a group of neurotoxins with a common tetrahydropurine backbone that block ion transport by voltage-dependent sodium-channels. The representative toxin of the PST class is STX, although more than 50 analogs have been described, some of them produced specifically by freshwater cyanobacteria [15]. DA and its, at least, 9 isomers (epi- and iso-domoic A–H) constitute the class of amnesic shellfish poisoning toxins (ASTs) [16]. DA binds to and activates kainate receptors, a subclass of glutamate receptors [17]. To protect human health, a few countries have established limits for the concentration of some freshwater toxins in drinking and recreational waters [18], mostly based on World Health Organization (WHO) guidelines that provisionally recommend as upper limit 1 mg of microcystin-LR (MC-LR) per liter of drinking

water [19]. The development of detection methods for freshwater toxins is necessary to comply with legal limits. Actually, many different methods have been developed for the detection of these toxins, including bioassays, molecular and analytical techniques, some of them allowing multi-detection [20–25]. The current trend in the aquatic toxins field is the development of inexpensive and efficient multiplexed screening methods in which the same sample is analyzed for the presence of multiple analytes in one assay [26]. Multi-detection screening methods save sample volume and hasten the acquisition of results reducing the number of analysis to be performed with confirmatory and/or quantitative methods. Flow-cytometry technology has been widely employed in clinical and research fields for the development of multiplexed assays in which different analytes present in the same sample are simultaneously detected [27]. Luminex technology uses microsphere classes with different spectral properties and surface carboxyl groups for covalent ligand attachment. Each microsphere class becomes specific for an analyte through the immobilization of a specific ligand. A fluidic system separates individual microspheres where respective red and green lasers distinguish microsphere class and quantify the attached compound. Multiplexing is provided by the incubation of a sample with multiple classes of analyte-specific microspheres simultaneously. In this work, a semi-quantitative microsphere-based multidetection method for five freshwater and brackish microalgal toxins has been developed using the Luminex technology. This method integrates previously developed assays for STX and DA [28,29] with newly developed assays for MC, CYN and ANA-a toxins into a multi-detection assay. The performance of this microspherebased method has been evaluated by comparison with ultra-performance liquid chromatography coupled to an ion trap-time of flight mass spectrometer (UPLC–IT-TOF-MS). 2. Material and methods 2.1. Materials Certified reference standard material of DA was obtained from CIFGA (Lugo, Spain). Certified reference standard material of STX dihydrochloride was obtained from the Institute for Marine Biosciences, National Research Council (Halifax, Canada). DA for immobilization was purchased from Merck Millipore (Darmstadt, Germany) and CIFGA (Lugo, Spain). ANA-a, CYN, MC-LR, MC-YR and MC-RR were obtained from ENZO (Farmingdale, NY). Biotin-a-bungarotoxin (a-BTX) was from Molecular Probes (Eugene, OR). Analytical standard of MC-LR, MC-YR and MC-RR, N-hydroxysuccinimide (NHS), sodium tetraborate decahydrate, jeffamine (2,20 -(ethylenedioxy)bis(ethylamine)), ethylenediamine,

Fig. 1. Structure of five common microalgal toxins: (A) MC-LR, (B) CYN, (C) ANA-a, (D) STX and (E) DA.

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boric acid, sodium phosphate monobasic, ethanolamine and Tween-20 were purchased from Sigma–Aldrich (Madrid, Spain). 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was purchased from Pierce (Rockford, IL). The anti-MC-LR (MC-LR-Ab), anti-CYN (CYN-Ab), anti-DA (DA-Ab) and antigonyautoxin 2/3 (GT-13A-Ab) antibodies were obtained as previously described [21,22,30,31]. The acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was purified and characterized as previously described [32,33]. The following selection of nine cyanobacterial strains from the LEGE (Laboratory of Ecotoxicology, Genomics and Evolution) Culture Collection (CIIMAR, Porto, Portugal) were cultured and lyophilized as described by Sanchez et al. [34]: Microcystis aeruginosa strains LEGE 00063, 05195 and 91341; Aphanizomenon ovalisporum strain LEGE X-001; Cylindrospermopsis raciborskii strains LEGE 97047 and 99043; Anabaena sp. strains LEGE X-002, 00233 and 00234. Phycoerythrin goat anti-mouse Ig antibody (PE-Ab) was purchased from Invitrogen (Eugene, OR), phycoLink streptavidin-R phycoerythrin (PE-SA) from Prozyme (Hayward, CA) and sodium azide from Fluka (Steinheim, Germany). Carboxylated microspheres (LC10019–01, LC10027–01, LC10038–01, LC10050–01 and LC10054–01) were from Luminex Corporation (Austin, TX). Luminex sheath fluid, multiscreen 96 well filter plates, 33 mm Millex filter with 0.22 mm pore size, 0.45 mm pore size ultrafreeMC (Durapore1 membrane) or ultrafree-CL centrifugal filters (low binding Durapore1 PVDF membrane) were purchased from Millipore (Billerica, MA). Acetonitrile and methanol were from high performance liquid chromatography (HPLC) grade and formic acid from American Chemical Society (ACS) grade. Reagent grade solvents and buffer constituents were employed. High purity water was from a Millipore Direct 8/16 water system (Billerica, MA). Phosphate-buffered saline solution (PBS) was 130 mM NaCl, 10 mM NaPO4, pH 7.4. PBS-BT solution was PBS supplemented with 0.1% w/v BSA and 0.1% v/v Tween-20. 2.2. Toxin or binding protein immobilization on the microsphere surface MC-LR, CYN, STX, DA and Ls-AChBP were covalently attached to the carboxylated surface of five different microsphere classes: LC10050-01, LC10019-01, LC10038-01, LC10054-01 and LC10027-01, respectively. The protocols for the immobilization of STX (National Research Council, Canada) and DA (Merck Millipore and CIFGA) were described in Fraga et al. [29]. The same protocols were followed with slight modifications for the attachment of CYN (ENZO) and MC-LR (ENZO). Briefly, for covalent binding of CYN, 2  106 microspheres were activated by adding 150 mL of 150 mg mL1 EDC and 23 mg mL1 NHS dissolved in water. After 30 min of incubation, this mixture was removed and 20% jeffamine in borate buffer (pH 8.5) was added for 1 h. After jeffamine removal, 41.5 mg free-CYN in 35 mL of H2O and 16 mL of 37% formaldehyde were added to pre-activated microspheres and allowed to react for 24 h. Subsequently, unbound toxin was removed and unreacted carboxyl groups were inactivated by 1 M ethanolamine-HCl during 30 min. For MC-LR immobilization carboxylated microspheres were activated using a solution of 154 mg mL1 EDC and 46 mg mL1 NHS. Later 1 M ethylenediamine in borate buffer (pH 8.5) was added for 1 h. The free NHS-ester groups were inactivated by 1 M ethanolamine-HCl for 20 min. Pre-activation of toxin involved 20 mg of free-MC-LR in 10 mL of DMSO diluted with 40 mL of 27 mg mL1 EDC and 12 mg mL1 NHS in 10 mM sodium acetate buffer (pH 4.5). Ethanolamine was removed and the pre-activated toxin was added to the microspheres and allowed to react for 4 h. The immobilization of Ls-AChBP was described in Rodríguez et al. [35]. At the end of each toxin immobilization, each microsphere class was washed with PBS and stored in PBS with 0.01% sodium

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azide at 4  C in the dark. To achieve maximum specific binding, the immobilized toxin-microspheres should be allowed to equilibrate for one week with at least one storage solution renewal. Ls-AChBP-microspheres were stored in PBS-BT without azide and used right away. Incubations were in the dark with constant shaking (700 rpm). 2.3. Microsphere-based multi-detection assay for MC-LR, CYN, ANA-a, STX and DA Five competition assays were performed simultaneously in the same well. The procedure started with a first incubation of 60 mL of the sample or calibration solution with 60 mL of the binding proteins (0.1 mg mL1 MC-LR-Ab, 1.3 pg mL1 CYN-Ab, 1 mg mL1 GT-13A-Ab, 0.6 mg mL1 DA-Ab and 3  103 pre-washed Ls-AChBPcoated microspheres in a filter plate well). After 1 h of incubation, 100 mL of this mixture was transferred to a second microtiter filter plate containing previously washed toxin-coated microspheres (2  103 MC-LR-microspheres + 2  103 CYN-microspheres + 2  103 STX-microspheres + 2  103 DA-microspheres). One hour later 100 mL of a-BTX (50 nM) was added for 30 min. Finally, following a thorough wash, 100 mL of PE-Ab (0.5 mg mL1) and PE-SA (4 mg mL1) were added for 1 h. After a washing step, the microspheres were suspended in 100 mL of PBS-BT. All the incubations were carried out in the dark with constant shaking (700 rpm). 2.4. Quantification of binding signals PE-fluorescence attached to the surface of the microspheres was quantified with a Luminex 200TM analyzer (LuminexCorp, Austin, TX). Microspheres were classified with a 635 nm laser and PE-fluorescence was quantified with a 532 nm laser. The acquisition volume was 75 ml and the minimum number for bead count was 100. 2.5. Extraction method for cyanobacterial samples Freeze-dried cyanobacterial strains were extracted with a simple procedure. 20 mg were reconstituted in 2 mL of 75% aqueous methanol, vortexed, sonicated (6  30 s) and centrifuged for 10 min at 3000  g at 20  C. The pellet was twice re-extracted with 2 mL 75% aqueous methanol and centrifugation. Supernatants were pooled together. This extract was further diluted in PBS-BT to appropriate toxin content and filtered (0.45 mm) for the microsphere-based assay. The same extracts were analyzed by UPLC–ITTOF-MS after their dilution in 10% aqueous methanol and filtration through a 0.22 mm filter. 2.6. Detection of MCs, CYN and ANA-a by UPLC–IT-TOF-MS An UPLC–IT-TOF-MS with an electrospray ionization (ESI) interface (Shimadzu, Kyoto, Japan) was used for the identification and quantification of MC-LR, MC-RR, MC-YR, CYN and ANA-a. UPLC separation was performed with an ACQUITY UPLC HSS T3 column (2.1 i.d. 100 mm, 1.8 mm particle size, 100 Å pore size, Waters, Milford, MA) coupled to an in-line filter kit. Mobile phases A and B were water and acetonitrile, respectively, both acidified with 0.1% formic acid. The UPLC flow rate was 0.45 mL min1 for a binary gradient of A and B, and temperature was maintained at 35  C. The gradient of mobile phase B was as follows: 5% for 1 min, 5–60% over 9 min, 80% for 1 min, 80–5% over 3 min and a final equilibration of the column at 5% for 5 min. All MSn were operated in positive mode with the following ESI source conditions: nebulizing gas flow, 1.5 L min1; curved desolvation-line and heat block temperature, 230  C; drying gas pressure, 100 kPa; and detector voltage, 1.65 kV.

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Each toxin was analyzed using MS1 and MS2 with ion accumulation times of 10 ms and 30 ms, respectively. For the quantification and identification of the toxins, the following transitions were employed (1.5 Da ion precursor width, quantification transitions in italics): 995.54 > 599.35 and 995.54 > 553.31 for MC-LR (collision energy (E) 26%, collision gas (Cg) 21%); 519.78 > 440.23 and 519.78 > 298.17 for MC-RR (E 22%, Cg 17%); 1045.52 > 599.35 and 1045.52 > 603.29 for MC-YR (E 25%, Cg 15%); 416.12 > 336.17 and 416.12 > 194.13 for CYN (E 20%, Cg 24%); 166.12 > 149.10 and 166.12 > 131.08 for ANA-a (E 14%, Cg 5%). Previously reported misidentification of ANA-a with phenylalanine [36] was avoided by chromatographic separation and fragment characterization of phenylalanine in these UPLC–IT-TOF-MS conditions. Calibration solutions for MC-LR, MC-RR, MC-YR, CYN and ANA-a were prepared from stock solutions of 1 mg L1 by further dilution in 10% aqueous methanol. Calibration levels ranged between 0.01 and 0.75 mg L1. The injection volume was 10 mL. Sodium trifluoroacetate was used for mass calibration. The limits of detection (LOD) and quantification (LOQ) were: 0.05 and 0.15 mg L1 for MC-LR, 0.03 and 0.08 mg L1 for MC-RR, 0.09 and 0.28 mg L1 for MC-YR, 0.04 and 0.12 mg L1 for CYN and 0.03 and 0.10 mg L1 for ANA-a, respectively. 2.7. Safety Toxins should be handled with gloves and eye protection at all times. Appropriate disposal methods should also be utilized. 2.8. Data analysis All experiments were performed in duplicate. The Student's t-test for unpaired data was used for statistical analysis. Calibration curves for the microsphere-based method were fitted using GraphPad Prism 5.0 by a four-parameter logistic equation obtained with a nonlinear regression fitting procedure: Y = Rhi + (Rlo  Rhi)/ (1 + 10^ ((Log IC50  X)  HillSlope)), where Rhi is the bottom or the response at infinite concentration, Rlo is the top or the response at 0 concentration, IC50 is the half maximal inhibitory concentration and X is the logarithm of concentration. Calibration curves and quantification of samples performed by UPLC–IT-TOF-MS were fitted using the LabSolutions LCMS software (Shimadzu, Kyoto, Japan). 3. Results and discussion 3.1. Optimization of five microsphere-based assays for the detection of freshwater and brackish toxins Five microsphere-based assays were individually optimized to detect MC-LR, CYN, ANA-a, STX and DA. Four inhibition immunoassays consisted of the competition of toxins attached to the

microspheres (MC-LR, CYN, STX or DA) with toxins in solution (sample or calibration solution) for binding to specific antibodies (MC-LR-Ab, CYN-Ab, GT-13A-Ab or DA-Ab). The ANA-a assay was based on the competition of ANA-a with biotin-labeled a-BTX for binding to the Ls-AChBP, previously immobilized on the microsphere surface. Specific antibody and a-BTX bound to the microspheres were finally quantified using PE-Ab and PE-SA. Optimization was based on adjusting the immobilization procedure, antibody/detector-protein dilutions and/or incubation times. Immobilization of a toxin or binding protein on the microsphere surface was the first optimization step. Immobilization results were evaluated with the Luminex system through the maximum binding signal (Max) displayed after contact with a specific antibody or a-BTX. STX and DA immobilization had been previously optimized for high Max [29] (Table 1). For MC-LR two different immobilization couplings, through the amine or carboxyl moieties of the toxin, were tested. Immobilization through carboxyl moieties provided a higher Max (Table 1) than through amine moieties and were used for further assay development. The protocol for the immobilization of CYN, being devoid of a carboxyl moiety, was similar to that one used for STX (Table 1). Finally, for the optimization of the Ls-AChBP attachment, concentrations from 0.32 to 6.4 mg mL1 were tested. Higher Max was achieved with 3.2 mg mL1 Ls-AChBP (Table 1). Optimization of single inhibition assays was done for MC-LR, CYN and ANA-a following the protocols for STX and DA previously published, and already included in a multi-detection assay for marine toxins [29]. With the final aim of multiplexing, some experimental conditions were maintained constant for the four immunoassays (MC-LR, CYN, STX and DA): 1 h incubation times and 0.5 mg mL1 PE-Ab concentration. In these conditions several primary antibody concentrations were tested for each toxin: 0.09–0.18 mg mL1 MC-LR-Ab, 0.95–1.9 pg mL1 CYN-Ab, 0.2–2 mg mL1 GT-13A-Ab and 0.02–1.9 mg mL1 DA-Ab. Binding signals and calibration curves were evaluated for adequate performance. Calibration solutions ranged from 0.01 to 50 mg L1 for MC-LR, from 0.004 to 20 mg L1 for CYN, from 0.3 to 3000 mg L1 for ANA-a, from 0.04 to 370 mg L1 for STX and from 0.003 to 310 mg L1 for DA. The final primary antibody concentrations, 0.1 mg mL1 MC-LR-Ab, 1.3 pg mL1 CYN-Ab, 1 mg mL1 GT-13A-Ab and 0.6 mg mL1 DA-Ab, were selected considering sufficient Max/non-specific binding signal (Min) ratio and the IC50 of the calibration curves (Fig. 2A, B, D and E, and Table 1). Finally, the Ls-AChBP-based assay optimized for spirolide detection [35] was tested for ANA-a detection (Fig. 2C and Table 1). To explore the assay capabilities in terms of sensitivity, longer incubation times and higher antibody dilutions were tested for MC-LR detection. Overnight incubations for the competition step were combined with lower antibody concentrations, ranging from 4 to 18 ng mL1. The highest sensitivity, with a dynamic range from 0.09 to 1.66 mg L1 and an IC50 of 0.3 mg L1, was provided by overnight incubation of free toxin (sample or calibration solution)

Table 1 Calibration curve parameters for MC-LR, CYN, ANA-a, STX and DA when using microsphere-based single- and multi-detection methods. Dynamic ranges (IC20–IC80), IC50, maximum (Max) and non-specific (Min) binding signals. Concentrations are expressed in mg L1, maximum and minimum binding signals in raw response units (RU) (mean  SEM, n = 3, *statistically significant difference from individual detection).

MC-LR CYN ANA-a STX DA

Individual Multiplex Individual Multiplex Individual Multiplex Individual Multiplex Individual Multiplex

IC20(mg L1)

IC50(mg L1)

IC80(mg L1)

Max, Min (RU)

0.6  0.2 0.6  0.1 0.7  0.1 0.7  0.1 24  5 30  4 1.4  0.2 1.6  0.3 1.4  0.3 1.9  0.2

1.8  0.2 1.9  0.1 1.4  0.2 1.3  0.1 93  11 61  4 * 4.2  0.4 5.4  0.4 5.1  1.1 4.9  0.9

5.5  0.1 6.2  0.4 2.7  0.2 2.6  0.3 389  20 130  9 * 15  0.3 25  7 18  3 13  4

1639  161, 44  2 1394  100, 38  2 350  20, 5  0.4 260  7*, 5  0.5 950  58, 86  2 2030  289*, 78  6 443  64, 4  0.4 363  30, 29  5* 2912  141, 25  6 2544  148, 26  6

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with 18 ng mL1 MC-LR-Ab, followed by 2 h incubation with MC-LR-coated microspheres (data not shown). 3.2. Optimization of the multi-detection microsphere-based assay Once the experimental protocol for individual detection was optimized, the five assays were combined in a multi-detection method. The final assay consisted of the incubation of five binding proteins with the calibration solution or sample, this mixture was later added to toxin-coated microspheres, and finally detected by the addition of a-BTX and the PE-labeled proteins. Individual and multi-detection assays were compared using calibration curves obtained simultaneously with calibration solutions containing a mixture of MC-LR, CYN, ANA-a, STX and DA (Fig. 2). Multidetection and single assays performed similarly (Fig. 2). The IC50, dynamic ranges (IC20–IC80), curve slopes, Max and Min for individual and multiplexed assays did not show statistically significant differences for MC-LR, CYN, STX and DA, except for CYN Max that was slightly lower in multi-detection (Fig. 2A, B, D and E, Table 1). The calibration curve obtained for ANA-a in multidetection was quite different from the curve obtained in the single detection assay showing a statistically different steepness of the slope, a reduction of the dynamic range and a slight increase of sensitivity (Fig. 2C, Table 1). The modification of ANA-a calibration curve in multi- versus single-detection might be due to non-specific antibody binding to the a-BTX which would be consistent with the significant increase of Max raw binding signals (Table 1). Although the characteristics of the assay change with multiplexing, as shown for other microsphere-based detection methods [37], the assay is still useful to detect ANA-a. LODs (estimated using the IC20, Table 1 [38,39]) of this multidetection microsphere-based method are close to previously published immunoassays [40]. Nowadays, provisional WHO recommendations establish that the upper limit of MC-LR in drinking water should be 1 mg L1 [19]. Considering that the LOD of the microsphere-based method for MC-LR is 0.6 mg L1, the assay would have enough sensitivity to detect MC-LR at the recommended limit. Additionally its sensitivity could be increased with longer incubations and lower antibody concentrations, lowering the LOD of the MC-LR assay to 0.09 mg L1. Although the sensitivity for MC-LR is adequate for current recommendations, future adjustments for all toxins may be necessary with the development of new regulations. 3.3. Assay cross-reactivity

Fig. 2. Effects of multiplexing on the calibration curves of MC-LR, CYN, ANA-a, STX and DA when using the microsphere-based Luminex method. Single- and multidetection calibration curves in buffer solution were obtained simultaneously for (A) MC-LR, (B) CYN, (C) ANA-a, (D) STX and (E) DA (mean  SEM, n = 3, *statistically significant difference from individual detection).

The cross-reactivity of the assay for MC-LR, MC-RR and MC-YR, three common MCs, was evaluated using the microsphere-based method. A calibration curve was obtained for each analog with calibration solutions prepared in PBS-BT. The calibration solutions were simultaneously assayed in separate wells using the MC detection protocol. The immunoassay provided similar calibration curves for the three toxins, with IC50 values of 1.20, 2.72 and 1.79 mg L1 for MC-LR, MC-RR and MC-YR, respectively (Fig. 3). The percentage of cross-reactivity of the MC-LR-Ab for the three analogs was calculated considering MC-LR the reference toxin: % cross reactivity (% CR) = (IC50 of MC-LR/IC50 of MC analog)  100. Cross-reactivity values for MC-RR and MC-YR were 44% and 67%, respectively (Fig. 3). Therefore, the MC assay can detect at least three analogs of this toxin class. Very little information is available about MCs relative toxicity. The estimated intraperitoneal LD50 values in mice for MC-LR, -RR and -YR (around 50, 300 and 110.6 mg kg1, respectively) indicate that MC-LR is the most potent of the three analogs [23,41]. Therefore, considering these data, the cross-reactivity of the MC-Ab used in this study is an acceptable match to the relative toxicity of these analogs. Actually,

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3.4. Comparative analysis of cyanobacterial strains by the microsphere-based assay and UPLC–IT-TOF-MS

Fig. 3. Cross-reactivity of MC-LR-immunoassay for MC-LR, MC-RR and MC-YR. (A) Calibration curves for MC-LR, MC-RR and MC-YR obtained simultaneously using the microsphere-based Luminex assay. (B) IC50, dynamic range (IC20–IC80) and percentage of cross reactivity for the three toxins. The percentage of crossreactivity was calculated considering MC-LR as reference toxin. The cross-reactivity is expressed in percentage and the concentrations in mg L1 (mean  SEM, n = 3).

the detection of several toxic analogs is an important feature of the assay, since 1998, WHO recommendations are provisional covering only MC-LR based on the lack of toxicological data on other microcystins [42]. This guideline will probably evolve in the near future to recommend limits of other microcystins, and other freshwater toxins, as it is already happening with legal regulations of several countries [4,18,42]. In regards to the other toxin groups, the multi-detection microsphere-based method is capable of detecting more than one analog per group. The GT-13A-Ab has been demonstrated to detect several PST analogs in marine samples, but poor cross-reactivity with N-1 hydroxy PST analogs was repeatedly reported [28,31,38,43]. STX, decarbamoyl (dc) STX, gonyautoxin (GTX) 2/3, GTX1/4, dcGTX2/3, neosaxitoxin (NEO), and C1/2, among others, have been found in freshwater ecosystems and their toxicities are assumed equal to the same compounds from marine origin [4,44]. Ideally, the cross-reactivity of the antibody should match the toxicity equivalency factor for all the toxins of the group. Unfortunately some of the N-1 hydroxy analogs, such as NEO, are among the more toxic PSTs. Toxicity evaluation by this assay would improve with an antibody specific for N-1 hydroxy PSTs. Sample screening with the current PST assay will require re-testing of negative samples by analytical methods, until a N-1 hydorxy PST assay is available. The previous characterization of the CYN-Ab indicated 2–11% cross-reactivity for deoxyCYN versus CYN [22]. DeoxyCYN did not show lethal toxicity by intraperitoneal administration in mice, contrary to CYN, which had an estimated LD50 of 200 mg kg1 [9,45]. Ls-AChBP binds to ANA-a and other toxins from different origins that could be detected with this assay, however none of them has been described in brackish or freshwaters to the extent of our knowledge [32,35,46]. Finally, there are no data that could provide information about the crossreactivity of DA and ANA-a assays for other members of these toxin classes. The individual assays compiled in the multiplexed technique were specific since none of them detected toxins of the other classes included in the multi-detection method.

Freeze-dried cyanobacterial biomass was extracted and analyzed by the microsphere-based assay and UPLC–IT-TOF-MS. A simple extraction method using aqueous methanol was developed to recover the five toxin classes and to warrant compatibility with protein-binding assays [28,29,35]. Aqueous methanol was used as extraction solution due to the hydrophilic nature of these toxins, and to avoid interferences generated by ethanol [29]. Preliminary tests using two different extraction solutions containing two ratios of methanol:water (1:3 and 3:1) showed that a higher content of methanol provided a slightly higher toxin recovery (data not shown). A cyanobacterial sample from a strain negative for the presence of these toxins (LEGE 233) was extracted for matrix interference tests. Several dilutions of LEGE 233 extract, 1:10, 1:100 and 1:1000 (v/v), were assayed with the multi-detection microsphere assay. Max and Min signals obtained with these sample dilutions were compared to signals obtained simultaneously in buffer. Dilution 1:10 (v/v) showed interferences with Max signal associated to CYN-, STX- and LsAChBPcoated microspheres (data not shown). Therefore, calibration curves of the five toxins were obtained simultaneously in buffer and in a 1:100 dilution of the LEGE 233 extract. The calibration curves demonstrated the lack of interference of cyanobacterial extract for the five assays (Fig. A.1). Cyanobacterial samples from in vitro cultures were assayed for the presence of the five toxin groups using the microsphere-based assay. All samples were assayed using a 1:100 dilution of the initial extract, however, positive samples were out of the linear range of the assay. Therefore, positive samples were re-assayed using dilutions of the extract ranging from 1:1000 to 1:10000. The same extracts were analyzed for MCs, CYN and ANA-a by UPLC–IT-TOF-MS (Fig. A.2–A.5). Both techniques were used to estimate the toxin contents of the nine strains (Table 2). The final results from the Luminex assay were reported as representative toxin equivalents. The estimated contents of MCs and CYN were similar for both methods (Table 2). Toxic and non-toxic strains from a same cyanobacterial species were discriminated by both Luminex and UPLC–IT-TOF techniques, although this LC–MS/MS (liquid chromatography–tandem mass spectrometry) method was not optimized for high sensitivity. The analysis of the strains positive for MC production by UPLC–IT-TOF-MS demonstrated the presence of MC-LR, MC-RR and MC-YR (Fig. A.3). These MCs were quantified using analytical standards and the amount of the three molecules was added to report the total content of MCs for comparison with the Luminex assay (Table 2). Quantification of ANA-a in LEGE X-002 was almost four times higher when using the microsphere-based assay than when using UPLC–IT-TOF-MS. The quantification by UPLC–IT-TOF-MS was done considering only ANA-a. However, the LEGE X-002 sample contained also a mass of m/z 182.12 (M + H+) consistent with the presence of epoxy-ANA-a. Its fragmentation (MS2) yielded transitions described for epoxy-ANA-a (Fig. A.5) [47]. The epoxy-ANA-a amount cannot be calculated due to the lack of analytical/certified standards. Additionally, the analysis of extracts from LEGE X-002 using a QTRAP LC/MS/MS instrument points to the production of three additional ANA-a analogs by this cyanobacterial strain [34]. Therefore, in these experimental conditions it is clear that the UPLC–IT-TOF-MS method underestimated the amount of ANA-a toxins. In addition to this demonstrated underestimation of ANA-a toxins by LC–MS, it is also possible that the Luminex assay overestimates the amount of ANA-a toxins. Cross-reactivity could not be determined for the members of this toxin class, and a higher affinity of some analogs for the Ls-AChBP would certainly yield an overestimation of toxin content when compared to ANA-a standard. This microsphere-based method can be considered a useful tool to detect the presence of cyanotoxins in cyanobacterial samples. The sensitivity, calculated considering our extraction protocol and

Please cite this article in press as: M. Fraga, et al., Multi-detection method for five common microalgal toxins based on the use of microspheres coupled to a flow-cytometry system, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.08.030

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Table 2 Quantification of MC, CYN and ANA-a in cyanobacterial samples by the multiplexed microsphere-based Luminex assay or UPLC–IT-TOF-MS. Nine cyanobacterial strains were assayed by the microsphere-based method for MC, CYN, ANA-a, DA and STX and analyzed by UPLC–IT-TOF-MS for MC-LR, MC-RR, MC-YR, CYN and ANA-a. Concentrations are expressed in mg of toxin per mg of biomass. For the Luminex assay toxin content is expressed as representative toxin equivalents (toxin eq.). For all samples the content of STX and DA by the Luminex assay was below the limit of detection (
mg tox per mg of biomass MCs

Cylindrospermopsis raciborskii LEGE 97047 Aphanizomenon ovalisporum LEGE X-001 Cylindrospermopsis raciborskii LEGE 99043 Microcystis aeruginosa LEGE 00063 Microcystis aeruginosa LEGE 05195 Microcystis aeruginosa LEGE 91341 Anabaena sp. LEGE X-002 Anabaena sp. LEGE 00233 Anabaena sp. LEGE 00234

CYN

ANA-a

Luminex MC-LR eq.

UPLC–IT-TOF-MS (MC-LR + MC-RR + MC-YR)

Luminex CYN eq.

UPLC–IT-TOF-MS

Luminex ANA-a eq.

UPLC–IT-TOF-MS



1.9  0.1 3.4  0.2
1.9  0.3 2.9  0.7


the minimum dilution compatible with the assay, was 0.014 mg mg1 of lyophilized cyanobacteria for MC-LR, 0.016 mg mg1 for CYN, 0.67 mg mg1 for ANA-a, 0.036 mg mg1 for STX and 0.043 mg mg1 for DA. Moreover, the multiplexed assay could also be used for the analysis of water samples from the field after optimization of a sample preparation method, which would require organic solvents and sonication for cell disruption and, possibly, minor concentration of the extract due to the high sensitivity of the assay. These results demonstrate that the multiplexed method would provide a cost-effective, rapid evaluation of the presence of five toxin classes. Each batch of 2  106 analyte specific microspheres allows the analysis of up to 400 samples in duplicates. The simultaneous estimation of the toxins content in 40 pre-extracted samples can be performed in 5 h. Sample number can be increased through the use of additional microtiter plates. Other options for multi-detection are analytical methods, such as LC–MS, which offer accurate quantification and identification of multiple toxin classes and analogs as far as certified reference standards are available [20,24,48]. For this work, UPLC–IT-TOF was selected in order to identify different analogs of these toxin classes even if analytical standards were not available. However, for freshwater toxin detection triple quadrupoles are more often used for LC–MS/MS detection, which provide higher sensitivities than TOF mass spectrometers [20,24,48,49], but the analysis of 40 samples in 5 h is not possible. Additionally, analytical methods require the use of expensive instrumentation and highly trained personnel, which increases the cost of sample analysis. Recently, a multiplexed method has been developed for the simultaneous immuno-detection of MC-LR, CYN and STX using a microfluidic chip [25], however, the detector device is not commercially available yet. 4. Conclusion These results demonstrate the performance of a microspherebased multi-detection assay as a semi-quantitative screening tool for the detection of freshwater/brackish toxins belonging to MC, CYN, ANA-a, PST and AST classes. This method would allow saving precious resources such as time and sample volume due to the simultaneous detection of five toxin classes. Additionally, this assay is easy-to-perform, rapid and suitable for microalgal samples with a previous simple extraction procedure. In fact, the

co-occurrence and apparent bioaccumulation of CYN and PST support the need for the development of multi-detection methods of freshwater microalgal toxins [50,51]. Conflict of interest The authors declare no competing financial interest. Acknowledgments The research leading to these results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01 and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016, and through Axencia Galega de Innovación, Spain, ITC-20133020 SINTOX, IN852A 2013/16-3 MYTIGAL. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD. From the European Union's Seventh Framework Programme managed by REA–Research Executive Agency (FP7/2007-2013) under grant agreement Nos. 265,409 mAQUA, 315,285 CIGUATOOLS and 312,184 PHARMASEA. The project MARBIOTECHNORTE-07–0124-FEDER-000047 and the Portuguese Governmental Foundation for Science and Technology (FCT) throught the projectsPesT-C/MAR/LA0015/2013. This research was partly funded from a European Sustainable Programme 2007–2013 under the European Regional Development Fund (“ERDF”) as the Advanced Asset Project. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.08.030. References [1] H.W. Paerl, R.S. Fulton, P.H. Moisander, J. Dyble, Harmful freshwater algal blooms, with an emphasis on cyanobacteria, Sci. World J. 1 (2001) 76–113. [2] Y. Kotaki, N. Lundholm, H. Onodera, K. Kobayashi, F.F.A. Bajarias, E.F. Furio, M. Iwataki, Y. Fukuyo, M. Kodama, Wide distribution of Nitzschia navisvaringica, a new domoic acid-producing benthic diatom found in Vietnam, Fisheries Sci. 70 (2004) 28–32. [3] H.W. Paerl, V.J. Paul, Climate change: links to global expansion of harmful cyanobacteria, Water Res. 46 (2012) 1349–1363.

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G Model ACA 233430 No. of Pages 8

8

M. Fraga et al. / Analytica Chimica Acta xxx (2014) xxx–xxx

[4] S. Merel, D. Walker, R. Chicana, S. Snyder, E. Baures, O. Thomas, State of knowledge and concerns on cyanobacterial blooms and cyanotoxins, Environ. Int. 59 (2013) 303–327. [5] W.W. Carmichael, V. Beasley, D.L. Bunner, J.N. Eloff, I. Falconer, P. Gorham, K. Harada, T. Krishnamurthy, M.J. Yu, R.E. Moore, et al., Naming of cyclic heptapeptide toxins of cyanobacteria (blue–green algae), Toxicon 26 (1988) 971–973. [6] L. Pearson, T. Mihali, M. Moffitt, R. Kellmann, B. Neilan, On the chemistry toxicology and genetics of the cyanobacterial toxins, microcystin, nodularin, saxitoxin and cylindrospermopsin, Mar. Drugs 8 (2010) 1650–1680. [7] S.R. Pereira, V.M. Vasconcelos, A. Antunes, Computational study of the covalent bonding of microcystins to cysteine residues-a reaction involved in the inhibition of the PPP family of protein phosphatases, FEBS J. 280 (2013) 674–680. [8] R. Banker, S. Carmeli, M. Werman, B. Teltsch, R. Porat, A. Sukenik, Uracil moiety is required for toxicity of the cyanobacterial hepatotoxin cylindrospermopsin, J. Toxicol. Environ. Health 62 (2001) 281–288. [9] R.L. Norris, G.K. Eaglesham, G. Pierens, G.R. Shaw, M.J. Smith, R.K. Chiswell, A.A. Seawright, M.R. Moore, Deoxycylindrospermopsin, an analog of cylindrospermopsin from Cylindrospermopsis raciborskii, Environ. Toxicol. 14 (1999) 163–165. [10] K. Terao, S. Ohmori, K. Igarashi, I. Ohtani, M.F. Watanabe, K.I. Harada, E. Ito, M. Watanabe, Electron microscopic studies on experimental poisoning in mice induced by cylindrospermopsin isolated from blue–green alga Umezakia natans, Toxicon 32 (1994) 833–843. [11] C.E. Spivak, B. Witkop, E.X. Albuquerque, Anatoxin-a: a novel potent agonist at the nicotinic receptor, Mol. Pharmacol. 18 (1980) 384–394. [12] P. Thomas, M. Stephens, G. Wilkie, M. Amar, G.G. Lunt, P. Whiting, T. Gallagher, E. Pereira, M. Alkondon, E.X. Albuquerque, et al., (+)-Anatoxin-a is a potent agonist at neuronal nicotinic acetylcholine receptors, J. Neurochem. 60 (1993) 2308–2311. [13] K.J. James, A. Furey, I.R. Sherlock, M.A. Stack, M. Twohig, F.B. Caudwell, O.M. Skulberg, Sensitive determination of anatoxin-a, homoanatoxin-a and their degradation products by liquid chromatography with fluorimetric detection, J. Chromatogr. A 798 (1998) 147–157. [14] D.K. Stevens, R.I. Krieger, Stability studies on the cyanobacterial nicotinic alkaloid anatoxin-a, Toxicon 29 (1991) 167–179. [15] M. Wiese, P.M. D'Agostino, T.K. Mihali, M.C. Moffitt, B.A. Neilan, Neurotoxic alkaloids: saxitoxin and its analogs, Mar. Drugs 8 (2010) 2185–2211. [16] J. Clayden, B. Read, K.R. Hebditch, Chemistry of domoic acid, isodomoic acids, and their analogues, Tetrahedron 61 (2005) 5713–5724. [17] J.T. Slevin, J.F. Collins, J.T. Coyle, Analogue interactions with the brain receptor labeled by [3H]kainic acid, Brain Res. 265 (1983) 169–172. [18] I. Chorus, in: Chorus I (Ed.), Current Approaches to Cyanotoxin Risk Assessment, Risk Management and Regulations in Different Countries, Federal Environment Agency (Umweltbundesamt), Dessau-Rosslau, 2012. [19] WHO, Guidelines for Drinking-water Quality. Addendum to Volume 2, Health Criteria and Other Supporting Information, second ed., WHO Publishing, Geneva, 1998. [20] J. Chen, T. Yan, J. Xu, S. He, P. Zhao, X. Yan, Simultaneous determination of toxins in algae and water samples by high-performance liquid chromatography with triple quadrupole mass spectrometry, J. Sep. Sci. 35 (2012) 1094–1101. [21] S. Devlin, J.P. Meneely, B. Greer, C. Greef, M.J. Lochhead, C.T. Elliott, Next generation planar waveguide detection of microcystins in freshwater and cyanobacterial extracts, utilising a novel lysis method for portable sample preparation and analysis, Anal. Chim. Acta 769 (2013) 108–113. [22] C.T. Elliott, C.H. Redshaw, K. George, First development and characterisation of polyclonal and monoclonal antibodies to the emerging fresh water toxin cylindrospermopsin, Harmful Algae 24 (2013) 10–19. [23] N. Gupta, S.C. Pant, R. Vijayaraghavan, P.V.L. Rao, Comparative toxicity evaluation of cyanobacterial cyclic peptide toxin microcystin variants (LR, RR YR) in mice, Toxicology 188 (2003) 285–296. [24] M. Maizels, W.L. Budde, A LC/MS method for the determination of cyanobacteria toxins in water, Anal. Chem. 76 (2004) 1342–1351. [25] J. Zhang, S. Liu, P. Yang, G. Sui, Rapid detection of algal toxins by microfluidic immunoassay, Lab Chip 11 (2011) 3516–3522. [26] N. Vilarino, M.C. Louzao, M. Fraga, L.P. Rodriguez, L.M. Botana, Innovative detection methods for aquatic algal toxins and their presence in the food chain, Anal. Bioanal. Chem. 405 (2013) 7719–7732. [27] J.S. Kim, F.S. Ligler, Utilization of microparticles in next-generation assays for microflow cytometers, Anal. Bioanal. Chem. 398 (2010) 2373–2382. [28] M. Fraga, N. Vilarino, M.C. Louzao, K. Campbell, C.T. Elliott, K. Kawatsu, M.R. Vieytes, L.M. Botana, Detection of paralytic shellfish toxins by a solid-phase inhibition immunoassay using a microsphere-flow cytometry system, Anal. Chem. 84 (2012) 4350–4356. [29] M. Fraga, N. Vilarino, M.C. Louzao, P. Rodriguez, K. Campbell, C.T. Elliott, L.M. Botana, Multidetection of paralytic, diarrheic, and amnesic shellfish toxins by

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40] [41]

[42] [43]

[44] [45]

[46]

[47]

[48]

[49]

[50]

[51]

an inhibition immunoassay using a microsphere-flow cytometry system, Anal. Chem. 85 (2013) 7794–7802. T.F. McGrath, K. Andersson, K. Campbell, T.L. Fodey, C.T. Elliott, Development of a rapid low cost fluorescent biosensor for the detection of food contaminants, Biosens. Bioelectron. 41 (2013) 96–102. K. Campbell, L.D. Stewart, G.J. Doucette, T.L. Fodey, S.A. Haughey, N. Vilarino, K. Kawatsu, C.T. Elliott, Assessment of specific binding proteins suitable for the detection of paralytic shellfish poisons using optical biosensor technology, Anal. Chem. 79 (2007) 5906–5914. S.B. Hansen, Z. Radic, T.T. Talley, B.E. Molles, T. Deerinck, I. Tsigelny, P. Taylor, Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein, J. Biol. Chem. 277 (2002) 41299–41302. S.B. Hansen, T.T. Talley, Z. Radic, P. Taylor, Structural and ligand recognition characteristics of an acetylcholine-binding protein from Aplysia californica, J. Biol. Chem. 279 (2004) 24197–24202. J.A. Sanchez, P. Otero, A. Alfonso, V. Ramos, V. Vasconcelos, R. Araoz, J. Molgo, M.R. Vieytes, L.M. Botana, Detection of anatoxin-a and three analogs in Anabaena spp. cultures: new fluorescence polarization assay and toxin profile by LC–MS/MS, Toxins (Basel) 6 (2014) 402–415. L.P. Rodriguez, N. Vilarino, J. Molgo, R. Araoz, M.C. Louzao, P. Taylor, T. Talley, L. M. Botana, Development of a solid-phase receptor-based assay for the detection of cyclic imines using a microsphere-flow cytometry system, Anal. Chem. 85 (2013) 2340–2347. M. Gugger, S. Lenoir, C. Berger, A. Ledreux, J.C. Druart, J.F. Humbert, C. Guette, C. Bernard, First report in a river in France of the benthic Cyanobacterium phormidium favosum producing anatoxin-a associated with dog neurotoxicosis, Toxicon 45 (2005) 919–928. J. Peters, M. Bienenmann-Ploum, T. de Rijk, W. Haasnoot, Development of a multiplex flow cytometric microsphere immunoassay for mycotoxins and evaluation of its application in feed, Mycotoxin Res. 27 (2011) 63–72. K. Campbell, S.A. Haughey, H. van den Top, H. van Egmond, N. Vilariño, L.M. Botana, C.T. Elliott, Single laboratory validation of a surface plasmon resonance biosensor screening method for paralytic shellfish poisoning toxins, Anal. Chem. 82 (2010) 2977–2988. E. Usleber, R. Dietrich, C. Burk, E. Schneider, E. Martlbauer, Immunoassay methods for paralytic shellfish poisoning toxins, J. AOAC Int. 84 (2001) 1649–1656. M. Weller, Immunoassays and biosensors for the detection of cyanobacterial toxins in water, Sensors 13 (2013) 15085–15112. Y.-M. Chen, T.-H. Lee, S.-J. Lee, H.-B. Huang, R. Huang, H.-N. Chou, Comparison of protein phosphatase inhibition activities and mouse toxicities of microcystins, Toxicon 47 (2006) 742–746. M.D. Burch, Effective doses, guidelines and regulations, Adv. Exp. Med. Biol. 619 (2008) 831–853. E.S. Fonfría, N. Vilariño, K. Campbell, C. Elliott, S.A. Haughey, B. Ben-Gigirey, J. M. Vieites, K. Kawatsu, L.M. Botana, Paralytic shellfish poisoning detection by surface plasmon resonance-based biosensors in shellfish matrixes, Anal. Chem. 79 (2007) 6303–6311. M.E. van Apeldoorn, H.P. van Egmond, G.J.A. Speijers, G.J.I. Bakker, Toxins of cyanobacteria, Mol. Nutr. Food Res. 51 (2007) 7–60. R. Li, W.W. Carmichael, S. Brittain, G.K. Eaglesham, G.R. Shaw, Y. Liu, M.M. Watanabe, First report of the cyanotoxins cylindrospermopsin and deoxycylindrospermopsin from Raphidiopsis curvata (cyanobacteria), J. Phycol. 37 (2001) 1121–1126. Y. Bourne, Z. Radic, R. Araoz, T.T. Talley, E. Benoit, D. Servent, P. Taylor, J. Molgo, P. Marchot, Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 6076–6081. K.J. James, J. Crowley, B. Hamilton, M. Lehane, O. Skulberg, A. Furey, Anatoxins and degradation products, determined using hybrid quadrupole time-of-flight and quadrupole ion-trap mass spectrometry: forensic investigations of cyanobacterial neurotoxin poisoning, Rapid Commun. Mass Spectrom. 19 (2005) 1167–1175. S.A. Oehrle, B. Southwell, J. Westrick, Detection of various freshwater cyanobacterial toxins using ultra-performance liquid chromatography tandem mass spectrometry, Toxicon 55 (2010) 965–972. M. Holcapek, R. Jirasko, M. Lisa, Recent developments in liquid chromatography–mass spectrometry related techniques, J. Chromatogr. A 1259 (2012) 3–15. J.P. Berry, A. Jaja-Chimedza, L. Davalos-Lind, O. Lind, Apparent bioaccumulation of cylindrospermopsin and paralytic shellfish toxins by finfish in lake Catemaco (Veracruz, Mexico), Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 29 (2012) 314–321. J.P. Berry, O. Lind, First evidence of paralytic shellfish toxins and cylindrospermopsin in a Mexican freshwater system, Lago Catemaco, and apparent bioaccumulation of the toxins in Tegogolo snails (Pomacea patula catemacensis), Toxicon 55 (2010) 930–938.

Please cite this article in press as: M. Fraga, et al., Multi-detection method for five common microalgal toxins based on the use of microspheres coupled to a flow-cytometry system, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.08.030