Separation and flow cytometry analysis of microplastics and nanoplastics

Abstract

In recent years, the utilization of flow cytometry for quantitative microplastic analysis has gained prominence. However, the current methods have some drawbacks that need to be improved. The present study aims to enhance the flow cytometry detection protocols for Nile red (NR) stained microplastics, facilitating distinct microplastic and nanoplastic enumeration. By elevating dimethyl sulfoxide (DMSO) concentration to 20%-30% within the solution, NR solubility improved and agglomeration reduced. The analysis of 26 replicates of polystyrene (PS) liquid samples through four distinct dot plots highlighted the superior accuracy of dot plots integrating yellow fluorescence. Through systematic staining of varying NR concentrations across three microplastic liquid samples (polyethylene terephthalate, polyethylene, and polypropylene), the optimal staining concentration was determined to be 15-20 μg/mL. The distributions of agglomerated NR and NR stained PS under two scenarios-dissolved NR and partially agglomerated NR-were compared. Results showed their distinct distributions within the side scatter versus yellow fluorescence dot plot. Counting results from gradient-diluted PS liquid samples revealed a microplastic detection lower limit of 10⁴ particles/mL, with an optimal concentration range of 10⁵-10⁶ particles/mL. Flow cytometric assessment of PS microspheres spanning 150 nm to 40 μm indicated a 150 nm particle size detection minimum. Our investigation validated the efficacy of NR staining and subsequent flow cytometry analysis across eleven types of microplastics. Separation and concentration of microplastics (1.0-50.0 μm) and nanoplastics (0.2-1.0 μm) were achieved via sequential sieving through 50, 1.0, and 0.2 μm filter membranes. We used a combination of multiple filtration steps and flow cytometry to analyze microplastics and nanoplastics in nine simulated water samples. Our results showed that the combined amount of microplastics (1.0-50.0 μm) and nanoplastics (0.2-1.0 μm) after filtration had a ratio of 0.80-1.19 compared to the total microplastic concentration before filtration. This result confirms the practicality of our approach. By enhancing flow cytometry-based microplastic and nanoplastic detection protocols, our study provides pivotal technical support for research concerning quantitative toxicity assessment of microplastic and nanoplastic pollution.

Keywords: Nile Red; counting region; flow cytometry; microplastics; multistage filtration; nanoplastics.