Counterstaining-assisted flow cytometry for small-sized microplastics detection and toxicity assessment released from teabags

The widespread presence of microplastics (MPs) in food products has raised increasing concern, while their accurate identification and quantification remain challenging, particularly for small-sized particles (< 100 μm). Teabags, often containing plastic filter membranes, represent an underestimated source of MPs exposure. In this study, a high-throughput flow cytometry-based method was developed for the rapid detection and quantification of small-sized MPs released from commercial teabags and brewed tea infusions. A counterstaining strategy that combines Calcofluor White and fluorescein isothiocyanate to label natural organic matter, together with Nile Red staining for MPs, effectively reduced background interference and enabled reliable quantification in tea infusions. Specifically, flow cytometric analysis showed that a single teabag released approximately 4.3 × 10 MPs during the first brewing, while secondary steeping markedly reduced particle abundance. Most MPs originated from the filter bag rather than tea leaves. Furthermore, cellular assays using RAW 264.7 macrophages and Caco-2 intestinal epithelial cells indicated that teabag-derived particles disrupted the expression of tight junction-associated molecules and induced inflammatory responses, suggesting potential adverse health implications compared with polystyrene microspheres of similar size. Overall, this study provides a rapid and sensitive analytical approach for quantifying small-sized MPs in food matrices, highlights potential gut health risks associated with plastic-containing teabags, and provides valuable insights for assessing MPs exposure and guiding food safety regulations.