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Ultrasensitive and high-throughput analysis of chlorophyll a in marine phytoplankton extracts using a fluorescence microplate reader

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Abstract

Chlorophyll a (Chl a) is the predominant pigment in every single photosynthesizing organism including phytoplankton and one of the most commonly measured water quality parameters. Various methods are available for Chl a analysis, but the majority of them are of limited throughput and require considerable effort and time from the operator. The present study describes a high-throughput, microplate-based fluorometric assay for rapid quantification of Chl a in phytoplankton extracts. Microplate sealing combined with ice cooling was proved an effective means for diminishing solvent evaporation during sample loading and minimized the analytical errors involved in Chl a measurements with a fluorescence microplate reader. A set of operating parameters (settling time, detector gain, sample volume) were also optimized to further improve the intensity and reproducibility of Chl a fluorescence signal. A quadratic regression model provided the best fit (r 2 = 0.9998) across the entire calibration range (0.05–240 pg μL−1). The method offered excellent intra- and interday precision (% RSD 2.2 to 11.2%) and accuracy (% relative error −3.8 to 13.8%), while it presented particularly low limits of detection (0.044 pg μL−1) and quantification (0.132 pg μL−1). The present assay was successfully applied on marine phytoplankton extracts, and the overall results were consistent (average % relative error −14.8%) with Chl a concentrations (including divinyl Chl a) measured by high-performance liquid chromatography (HPLC). More importantly, the microplate-based method allowed the analysis of 96 samples/standards within a few minutes, instead of hours or days, when using a traditional cuvette-based fluorometer or an HPLC system.

TChl a concentrations (i.e. sum of Chl a and divinyl Chl a in ng L-1) measured in seawater samples by HPLC and fluorescence microplate reader.

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Acknowledgements

The study was financially supported by a mobility grant under the EU Erasmus+ Programme for Higher Education, as well as from the EU-FP7 SeaBioTech project (Grant Number 311932). We thank the HCMR POSEIDON system team for providing the seawater samples from the E1-M3A site.

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Authors and Affiliations

  1. Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, 71500, Gournes, Heraklion, Crete, Greece

    Manolis Mandalakis & Paraskevi Polymenakou

  2. Department of Chemistry, Vilnius University, 24 Naugarduko, 03225, Vilnius, Lithuania

    Austėja Stravinskaitė

  3. Institute of Oceanography, Hellenic Centre for Marine Research, 71500, Gournes, Heraklion, Crete, Greece

    Anna Lagaria & Stella Psarra

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  1. Manolis Mandalakis
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  2. Austėja Stravinskaitė
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  3. Anna Lagaria
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  4. Stella Psarra
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  5. Paraskevi Polymenakou
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Correspondence to Manolis Mandalakis.

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Mandalakis, M., Stravinskaitė, A., Lagaria, A. et al. Ultrasensitive and high-throughput analysis of chlorophyll a in marine phytoplankton extracts using a fluorescence microplate reader. Anal Bioanal Chem 409, 4539–4549 (2017). https://doi.org/10.1007/s00216-017-0392-9

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  • DOI: https://doi.org/10.1007/s00216-017-0392-9

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