Separation and flow cytometry analysis of microplastics and nanoplastics

Area of Science:

• Environmental Science
• Analytical Chemistry
• Toxicology

Background:

• Flow cytometry is increasingly used for microplastic analysis.
• Current methods require improvement for accurate microplastic and nanoplastic enumeration.
• Nile red (NR) staining is a common technique, but its efficiency can be enhanced.

Purpose of the Study:

• To optimize flow cytometry protocols for enhanced detection of Nile red-stained microplastics and nanoplastics.
• To improve the accuracy and sensitivity of microplastic and nanoplastic quantification.
• To provide a robust method for assessing microplastic and nanoplastic pollution.

Main Methods:

• Optimized Nile red staining protocols by adjusting dimethyl sulfoxide (DMSO) concentration.
• Utilized specific dot plots (side scatter vs. yellow fluorescence) for accurate enumeration.
• Determined optimal NR staining concentration (15-20 μg/mL) and flow cytometry parameters.
• Employed sequential filtration for microplastic and nanoplastic separation and concentration.
• Validated the method across eleven microplastic types and in simulated water samples.

Main Results:

• Increased DMSO concentration (20%-30%) improved NR solubility and reduced agglomeration.
• Optimal NR staining concentration determined to be 15-20 μg/mL.
• Microplastic detection lower limit established at 10^4 particles/mL, with an optimal range of 10^5-10^6 particles/mL.
• Minimum detectable particle size for flow cytometry was 150 nm.
• Filtration and flow cytometry method showed high recovery rates (0.80-1.19) for microplastics and nanoplastics in simulated samples.

Conclusions:

• The enhanced flow cytometry protocol provides accurate and sensitive quantification of microplastics and nanoplastics.
• The optimized method is effective for diverse microplastic types and environmental matrices.
• This approach offers crucial technical support for microplastic and nanoplastic toxicity assessments.