Elsevier

Talanta

Volume 187, 1 September 2018, Pages 47-58
Talanta

Lipophilic marine biotoxins SERS sensing in solutions and in mussel tissue

https://doi.org/10.1016/j.talanta.2018.05.006Get rights and content

Highlights

  • Multi-laser, micro-SERS analysis of three lipophilic biotoxins from the okadaic acid group and yessotoxin.

  • Adsorption mechanism of the OA group species with respect to the AgNPs.

  • Micro-SERS signalling of edible mussel tissue.

  • SERS translation from the lab to the field.

Abstract

To detect and recognise three structurally related marine biotoxins responsible for the diarrheic shellfish poisoning (DSP) symptom, namely okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) respectively, as well as the structurally different yessotoxin (YTX), we developed a novel surface-enhanced micro-Raman scattering (micro-SERS) approach to investigate for the first time their micro-SERS signalling in solution and jointly analysed them in conjunction with the normal and toxic mussel tissue. YTX provided the main SERS feature surprisingly similar to DTX-1 and DTX-2, suggesting similar molecular adsorption mechanism with respect to the AgNPs. A fingerprint SERS band at 1017 cm−1 characteristic for the C-CH3 stretching in DTX-1 and DTX-2 and absent in OA SERS signal, allowed direct SERS discrimination of DTX-1,2 from OA. In acid form or as dissolved potassium salt, OA showed reproducible SERS feature for 0.81 μM to 84.6 nM concentrations respectively, while its ammonium salt slightly changed the overall SERS signature. The inherently strong fluorescence of the shellfish tissue, which hampers Raman spectroscopy analysis, further increases when toxins are present in tissue. Through SERS, tissue fluorescence is partially quenched. Artificially intoxicated mussel tissue with DSP toxins and incubated with AgNPs allowed direct SERS evidence of the toxin presence, opening a novel avenue for the in situ shellfish tracking and warning via micro-SERS. Natural toxic tissue containing 57.91 μg kg−1 YTX (LC-MS confirmed) was micro-SERS assessed to validate the new algorithm for toxins detection. We showed that a portable Raman system was able to reproduce the lab-based SERS results, being suitable for in situ raw seafood screening. The new approach provides an attractive, faster, effective and low-cost alternative for seafood screening, with economic, touristic and sustainable impact in aquaculture, fisheries, seafood industry and consumer trust.

Introduction

Marine biotoxins accumulation in seafood products raises a serious risk to consumer's health and may ban the specific economic activity when intoxication suspicious cases occur. To date, the commonly used techniques to control the presence of harmful marine biotoxins in seawater or seafood products are mainly based on HPLC [1], LC-MS/MS [2], usually coupled with UV or florescent detection, bioassays [3], or immunoassays with biosensors [4] and more recent, analytical biosensing methods [5], [6], [7], [8]. Although sensitive, the main drawback of the conventionally agreed techniques for toxins detection and monitoring in perishable seafood is that they cannot provide prompt answer on the seafood product status and toxins presence because of the multi-steps protocols required. The delayed results may pose negative impact on the seafood farming sector and consumer trust. A significant issue is the lack of portability since current techniques rely on laboratory-based complex procedures. McNamee et al. (2016) [5] suggested that surface plasmon resonance (SPR) biosensing could provide more sensitive description of the algal bloom events and highlighted the advantages and drawback of the methods existing so far. None of the reviewed techniques is portable.

Among the marine biotoxins increasingly detected in seafood, okadaic acid (OA) is the most frequent [9]. Along with the structurally related dinophysistoxins (DTX-1, DTX-2, and DTX-3), OA is released in the food web by harmful marine dinoflagellates belonging to the genus Dinophysis and Prorocentrum and accumulate in the gills, digestive gland and edible tissue of various seafood products and further in consumers, being responsible for the diarrheic shellfish poisoning (DSP) prominent syndrome [9], [10]. Yessotoxin (YTX), a lipophilic, sulfur bearing polyether biotoxin, was linked to inflammation of the intestinal tract, sodium release, passive fluid loss and diarrhea in humans [10], [11], [12], [13], [14]. Concerning the structurally different yessotoxins (YTXs), and their simultaneous presence along with the DSP responsible species, Paz et al. [11] provided a comprehensive review on their origin, occurrence, toxicological properties and toxicity and highlighted the wide variability in the profile and the relative abundance of YTXs in bivalves and dinoflagellates as well as the synergistic toxic effect when many toxin species naturally co-exist. Additional research for improving optical or electrochemical output of toxins biosensing platforms [15] is certainly needed for understanding tissue signalling and emerging properties when toxic species are accumulated.

Regarding the quantitative determinations, the highest concentrations of OA (21.5 ng g−1) and DTX1 (8.4 ng g−1) were detected in Korean gastropods using HPLC, while the most recent surface plasmon resonance (SPR)–based immunosensing method [16] reported for OA 2.6 μg L−1, equivalent to 12 μg of OA per one kg mussel meat. Quantitative determination of OA toxin in commercially harvested shellfish [17] based on the luminescence of the lanthanide nanoparticles with fluorophore-labelled antibody binding OA showed a linear response in the 0.37–3.97 μM OA range with a detection limit of 0.25 μM. Further, using a luminescence resonance energy transfer (LRET)-based concept, Stipić et al. [18] developed a sensing method to indirectly detect the toxin based on the antigen-antibody recognition. Other methods, including electrospray ionization (ESI) liquid chromatography-mass spectrometry (LC-MS) [19] reported OA detection at 7 µg kg−1 shellfish meat (SM).

The Commission Regulation (EU) No. 15/2011 [20] has agreed the LC–MS/MS as the reference method for the determination of DSP toxins in shellfish (European Parliament, Council of the European Union, 2011 [20]). The maximum level in seafood products admitted by regulations is 160 μg of OA per kilogram (0.16 ppm) for okadaic acid, dinophysistoxins and pectenotoxins in combination, and 1 mg YTX per kilogram for yessotoxins (European Food Safety Authority, EFSA, 2010 [21]). FDA approved 0.2 ppm okadaic acid + DTX-1 (FDA and EPA Safety Levels in Regulations and Guidance, [22]).

As recently stated [23], there is a gap in the techniques available for risk assessment and bioavailability of the toxins from seafood to digestive tract and blood stream. Structurally related polyether lipophilic toxins responsible for the DSP syndrome yet appear difficult for real time monitoring and new effective methods are highly desired.

In response to the need for faster, sensitive, cheaper and reliable methods for toxins detection and seafood monitoring in situ, we adapted surface enhanced micro-Raman scattering (SERS) with silver nanoparticles (AgNPs) to assess the detection capability of the technique for extremely limited amount of lipophilic toxin solutions and to get insight into the molecular adsorption mechanism. SERS technique, although not new, currently expands in broaden area of novel approaches [24], [25], [26], [27], on one hand due to the development of nanotechnologies capable of producing plasmonic nanoparticles with controlled properties, and on the other hand, due to the availability on the market of flexible, sensitive, compact and portable Raman systems. Our previous reports [25], [26], [27] expanded the SERS applicability towards aquaculture and marine field. Apparently, SERS would provide substantial advantages in terms of sensitivity, portability for in situ analyses, rapidity and relative low cost compared to the current agreed techniques, provided that the signal detection, recognition and shellfish tissue diagnostic algorithm is achieved. The prospect for SERS translation to field applications is currently hampered by the lack of lipophilic toxins robust vibrational database. Moreover, Raman spectroscopy data of soft shellfish tissue are scant to absent.

Here, the SERS response of three toxins from OA group, namely okadaic acid (OA), dinophysistoxin-1 (DTX-1), dinophysistoxin-2 (DTX-2) and the title compound from yessotoxins group (YTX), was investigated in the presence of AgNPs, to assess: (i) the characteristic SERS signal of lipophilic toxins to consolidate a spectral database; (ii) to understand their molecular adsorption mechanism; and (iii) to further employ their SERS signalling in extended experiments on edible shellfish tissue to probe the toxins micro-SERS sensing. To achieve the latter, consistent Raman spectroscopy analysis of soft shellfish tissue in various normal or toxic forms is reported for the first time.

Section snippets

Chemicals

Three different stocks of okadaic acid were used: OA ammonium salt from Prorocentrum concavum, (≥ 90% (HPLC), solid (Sigma) 10 μg,); OA potassium salt, (Alfa Aesar, > 98%, 300 μg, product Nr. J61791, lot U08A024) and certified calibration solution of OA (CRM-OA-c, lot 20070328), in methanol with 0.1 mM acetic acid of concentrations 13.7 ± 0.6 µg mL−1 (17 μM) at + 20 °C.

Certified calibration solutions of DTX-1, DTX-2 and YTX included in the List of Okadaic Acid Group Toxins Reference Materials

Lab-based Raman systems

Raman and SERS spectra were recorded with a confocal Renishaw InVia Reflex Raman microscope, using the 532 nm or 785 nm laser lines for excitation. For toxins data acquisition, 5 accumulations × 5 s at progressively increased laser power from 1 to 100 mW were applied to avoid excessive sample heating and/or photodecomposition. Spectral acquisition was achieved employing the 20 × or 100 × objective adapted to the 90° laser deviation toward a liquid holder accessory and the laser focus was

Results and discussions

Raman scattering signal of bulk colloidal nanoparticles showed no bands except water, in agreement with previous reports [25], [26], [27], while the absorbance maximum confirmed the reproducibility robustness of the classical wet synthesis. Freshly obtained LM-AgNPs showed absorbance maximum at 406 nm with full width at half maximum (FWHM) of 96 nm, while HY-AgNPs at 418 nm and FWHM of 140 nm, respectively. Both the LM-AgNPs and HY-AgNPs revealed optical and morphological properties consistent

Outlook

Concerning the toxins accumulation in seafood edible tissue, Ehara et al. (2015) [35] elucidated the crystal structure of okadaic acid binding protein 2.1 (OABP2.1) isolated from Halichondria okadai sponge. OABP2.1 showed strong affinity for OA and its structure revealed two α-helical domains with the OA molecule deeply buried inside the protein. Linking this information with the Raman spectroscopy technique based on the inelastic light scattering by molecules, it appears challenging to detect

Conclusions

We investigated the interface between Ag nanoparticles (AgNPs) and lipophilic marine toxins exploiting an adapted micro-SERS technique for extremely limited toxin samples. A fingerprint SERS band at 1017 cm−1 characteristic for the C-CH3 stretching in DTX-1 and DTX-2 and absent in OA SERS signal, allowed direct SERS discrimination of DTX-1,2 from OA. In acid form or as dissolved potassium salt, OA showed reproducible SERS feature for 0.81 μM to 84.6 nM concentrations respectively, while its

Acknowledgements

This paper has been prepared as a part of the project acronym JADRANSERS which has received funding through NEWFELPRO project (2013–2017) grant rr. 5 under grant agreement nr. 291823 MSE, Marie Curie FP7-PEOPLE-2011-COFUND. Cs. M acknowledges the financial support of the Sectorial Operational Program for Human Resources Development 2007–2013, co-financed by the European Social Fund, under the project number POSDRU/159/1.5/S/132400. The Babeş-Bolyai University Research Infrastructure financed by

Conflict of interest disclosure

Declarations of interest: none.

Statements about author contributions

All authors extensively contributed to the work presented in this paper. S. C. P. designed and performed experiments, analysed data and wrote the paper, Cs. M., U. I., M. M. V. B. G. performed experimental parts and contributed to the data analysis and manuscript writing; V. C. contributed to the data analysis and interpretation. B.G. contributed with the mussel & aquaculture expertise, experimental approach with the soft mussel tissue, manuscript writing. All

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