Optimization of Fluorescent Staining for Micro- and Nanoplastic Identification in Flow Cytometry

This experimental study investigates the critical factors affecting fluorescent staining and detection of micro and nanoplastics (MNPs) in environmental aqueous matrices using flow cytometry (FCM). While FCM is still a relatively novel approach in this field, it offers great potential for detecting and quantifying small particles—regardless of polymer type, shape, or concentration. The staining of MNPs with fluorescent dyes improves their identification in complex environmental samples, while co-staining enables the discrimination of plastics from biological particles. Many of these dyes can label cellular components (e.g., nucleic acids, mitochondria, or membranes), which may interfere with the specific polymer's detection. Additionally, optimizing dye concentration is essential, as excessive amounts can lead to aggregation and precipitation of the unabsorbed dye, possibly interfering with the identification of plastics through false positives. Finally, selecting an appropriate solvent is crucial to maintaining dye solubility and ensuring reliable FCM analysis. This study investigates the key factors that influence MNP labelling and the enhancement of the fluorescent signals in FCM analysis. Specifically, we leverage the lipophilic dye Nile Red (NR) to identify otherwise untraceable, not coloured and hydrophobic MNPs. We investigated the effect of increasing the ratio of the polar solvent dimethyl sulfoxide (DMSO) on NR solubility and the efficacy of a filtration step before staining to stic particle detection via FCM. The applied FCM approach solved aspects such as: (i) the increased background due to NR presence, (ii) the maximum emission depending on the solvatochromic dye (695/40 BL3 for NR), and (iii) the optimal threshold on fluorescence, as scattering signals alone do not adequately resolve MNPs in environmental matrices. Optimal conditions were determined by analysing NR-stained MNP populations in the method was tested using beads of different diameters (1.0 and 3.0 μm) in environmental matrices such as wastewater effluent. Further investigation explored the potential of three additional fluorochromes—Acridine Orange, Neutral Red, and New Methylene Blue N—by analysing their signals and identifying optimal emission filters. The proposed single-cell FCM analysis demonstrated strong potential for precise discrimination and quantification of micro and nanoplastics with high sensitivity and rapid processing.