I ntensity, frequency and geographic distribution of harmful algae blooms have increased also in areas previously unharmed (Dubois et al., 2010) like the Northern part of the Mediterranean Sea. The toxins produced by these microrganisms can contaminate seafood and represent a potential threat to human health. O ne of those threats is the Paralytic Shellfish Poisoning (PSP) due to non-proteinaceous paralytic neurotoxins (Tetrodotoxins TTXs) , that selectively bind to voltage-gated sodium (Na+) channels interfering with or completely inhibiting the neural transmission in nerve and muscle tissues (Denac et al., 2000; Magarlamov et al., 2017) . TTX is commonly present in Tetraodontidae Teleosts, anyway it has been recently reported in several shellfish species. The European Food Safety Authority (EFSA) has specifi ed a concentrations of 44 μg of TTX or TTX analogues equivalents per kg of shellfish meat a s maximum theshold for food safety (Knutsen et al., 2017) . T he elective methods for TTX detections are based on chromatography separations mainly in tandem with MS like LC/MS(/MS) with hydrophilic interaction liquid chromatography (HILIC) or MS/MS and the method of extraction is optimized (Turner et al., 2023) with high sensitivity and low concentrations of TTX (from µg/ml to ng/ml). Anyway the need to expand monitoring plans are requiring analytical efforts to perform an early diagnosis, with a minimal approach also for sample preparation. To meet such requirement for a rapid preliminary screening, an immunsensor assay , based on fluorescence detection, coupled with PocOrEl (Orel d.o.o, SI) analytical devices for the detection of TTX in mussel meat is presented.
Material and Methods
A calibration curve was prepared with TTX (Creative Diagnostic Inc., USA) in buffer acetate and diluted with PBS at a ratio of 1:10 to obtain samples with 80, 60, 40, 20 and 0 µ g/L of TTX; data were compared with mussels’ meat spiked with the same amount of TTX to obtain the same absolute TTX concentration. T he effect of the matrix in the assay that retrace an indirect ELISA test was evaluated on mussel extract after or without a filtration step through 20µm filters . A black plastic cartridge with a reaction chamber of 35 μl volume has been used (Daniso et al., 2020) for immunosensor implementation . The bottom of the reaction chamber is a polyethylene (PE) transparent substrate subjected to a coating with selected organosilane (GPTES) for better adhe sion of the TTX-BSA molecule (Daniso et al., 2021) . The TTX-BSA (Creative Diagnostic Inc., USA) mix was deposited in the reaction chamber and let to react overnight at room temperature. Subsequently, a blocking step with defatted milk 2% was performed. Then, sample mix, composed of sample and MonoclonalAntibody-TTX (1.5 mg/ml) diluted 1:250 in PBS, was loaded in the well and was incubate at 37°C per 15 min. Afterward, the slides were washed twice to eliminate unbound MAb-TTX to the substrate and 40µl of conjugated PoliclonalAntibody with the florophore Atto 430LS (2 ml/ml ) diluted 1:50 was added to the well and incubate d as above . After washing twice, the cartridge was left to dry before measurements with PocOrEl (Orel d.o.o) analytical devices. Data were validated with an ELISA assay using a Sunrise (Tecan USA).
The trend of the obtained results for the two sets of samples follows the expectation of a decrease in the signal with the increase of the toxin concentration of sample, and the signals in the two situations are almost equivalent. The analysis carried out with the TTX in PBS provided similar signals in comparison with the data obtained for the spiked mussels samples that contain equal amount of target toxin. The filtration step with a 20 µ m filter of spiked homogenized mussels and the dilution of 1:10 of the Acetate buffer used for the extraction resulted a key step. A reduction of the Mab-TTX activity was observed when the raw homogenized mussel meat was used while the filtration step at 20µm reduce the interference of the matrix with the Mab-TTX . The cut-off value of 44 µ g/kg of the analysis is marked on the graph with the red arrow ; samples that exhibit a number of counts close to the corresponding cut-off figure should be considered as positive.
In conclusion, the presented implemented assay for the Poc-Orel system (OrEl d.o.o , SI) shows a good discrimination between positive and negative sample even if must be refined , at the moment is limitated at the detection of TTX and should be implemented for the analogues compound in order to meet the requirements of extensive monitoring applications.
Acknowledgment: The study was funded by TOXIPOC project, Regione Friuli Venezia Giulia (POR FESR 2014-2020. A 1.3.b. DGR n. 1489/2017
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Magarlamov et al. (2017). https://doi.org/10.3390/toxins9050166
Turner et al.(2023). https://doi.org/10.1093/jaoacint/qsad006