Plastibodies for Multiplexed Detection and Sorting of Microplastic Particles in High-throughput

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Wiwik Bauten, Maximilian Nöth, Tetiana Kurkina, Francisca Contreras, Yu Ji, Cloé Desmet, Miguel-Ángel Serra, Douglas Gilliland, Ulrich Schwaneberg., Science of the Total Environment, doi.org/10.1016/j.scitotenv.2022.160450

Combination of fluorescent polymer-binding peptides (plastibodies) labeling and flow cytometry enables multiplexed quantification and sorting of stained microplastic particles in high-throughput showing high efficiency and accuracy (>90%).

Potential health and ecological risks associated with microplastic contamination demand the development of novel analytics to quantify and sort microplastic particles in aqueous suspensions. Sorting of microplastic would allow subsequent quantification of toxic compounds entrapped in different types of microplastic, through complementary techniques, which would enable the determination of health risks in a material-specific manner. Material-binding peptides (MBPs) like anchor peptides are material-specific and strongly bind to synthetic polymers such as polystyrene, polypropylene, polyethylene terephthalate, and polyurethane, which can be used as a biotechnological tool to tag microplastic. In the present study, we report a method for quantification of microplastic based on MBPs LCIF16C (engineered MBPs Liquid Chromatography Peak I by substituting the amino acid Phe16 with Cys) conjugated to three Alexa-fluorophores (AF488, AF594, and AF647) generating three fluorescent material-binding peptides (also termed plastibodies) (LCIF16C-AF488, LCIF16C-AF594, and LCIF16C-AF647), and followed by Flow cytometry by fluorescence-activated cell sorting (FACS) analysis. The accurate differentiation of labeled and non-labeled particles by flow cytometry (Chi Squared Test, p < 0.01) confirms the chosen labeling strategy efficiency and verifies the power of flow cytometry for the quantification of microplastic in diverse mixtures. Mixtures of polystyrene microplastic that varied in size (500 nm to 5 µm) and varied in labeled populations were analyzed and sorted into distinct populations reaching sorting efficiencies >90 % for 1x10⁶ sorted events. Finally, a multiplexed quantification and sorting with up to three plastibodies was successfully achieved to validate that the combination of plastibodies and flow cytometry is a powerful and generally applicable methodology for multiplexed analysis, quantification, and sorting of microplastic particles.

We gratefully acknowledge the "BioProMare: PLASTISEA" (Harvesting the marine Plastisphere for novel cleaning concepts) project; [FKZ: 031B0867E]) and BioökonomieREVIER_INNO PlastiQuant (FKZ: 031B0918E), the German Federal Ministry of Education and Research (BMBF) for financial support.

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Wiwik Bauten, Maximilian Nöth, Tetiana Kurkina, Francisca Contreras, Yu Ji, Cloé Desmet, Miguel-Ángel Serra, Douglas Gilliland, Ulrich Schwaneberg., Science of the Total Environment, doi.org/10.1016/j.scitotenv.2022.160450

  Copyright: © Science of the Total Environment Schematic illustration of Plastibodies-mediated fluorescent labeling enables high-throughput multiplex quantification and sorting of microplastic particles using flow cytometry.