Aquaculture Europe 2023

September 18 - 21, 2023


Add To Calendar 20/09/2023 11:00:0020/09/2023 11:15:00Europe/ViennaAquaculture Europe 2023PERPECTIVES OF AN IMMUNOBIOSENSOR FOR THE TETRODOTOXIN DETECTION IN MUSSELSSchubert 4The European Aquaculture Societywebmaster@aquaeas.orgfalseDD/MM/YYYYaaVZHLXMfzTRLzDrHmAi181982


E. Daniso *1, S. Susmel1 , P. Melpignano2 , F. Tulli1.


1Università degli Studi di Udine, Dipartimento Scienze Agroalimentari, Ambientali e Animali, via Sondrio 2/A, 33100 Udine (IT)

2OR-EL d.o.o., Volariceva Ulica 6, 5222, Kobarid, Slovenia

E -mail:



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 


Daniso et al. (2021).

Daniso et al. (2020).

Denac et al. (2000).

Dubois et al. (2010).

Knutsen et al. (2017).

Magarlamov et al. (2017).

 Turner et al.(2023).