Microplastics and nanoplastics detection using flow cytometry: Challenges and methodological advances with fluorescent dye application

Abstract

Flow cytometry (FC) enables the precise quantification of specific types of microparticles and larger nanoparticles (>200 nm) in liquid media. Initially developed for biological applications, this technique has recently been adapted to the environmental field for the measurement of microplastics and nanoplastics (MNPs). Nile Red, a fluorochrome extensively used in MNP analysis due to its effectiveness and accessibility, has been applied to significantly enhance the sensitivity and specificity of MNP detection of this technique. Additionally, flow cytometry offers the advantage of automated detection, allowing the quantification of smaller particles, including those under 1 µm, which are often missed by traditional spectroscopic methods. However, despite its promise, the presence of undissolved dye in aqueous media presents a significant challenge for accurate quantification. In recent years, various methodologies have been developed to overcome these limitations, including the use of co-solvents, surfactants, and pre-filtration or pre-sonication techniques to enhance quantification accuracy. This review examines recent literature on MNPs detection via FC, with a focus on technical improvements made and the remaining metrological challenges, offering insights into how this method can be further refined for future investigations.

Keywords: Flow cytometry; Microplastic; Nanoplastic; Nile red.

Graphical abstract

Fig. 1

Schematic representation of the flow cytometer with the alignment of particles, the diffusion parameters (FSC and SSC) and the fluorescence parameter measurements.

Fig. 2

Example of singlet gating based on FSC area signal, illustrated on a Dot-Plot of SSC peak area versus FSC peak area obtained using calibration beads, adapted from [32].

Fig. 3

Example of parameter and gating calibration with side scatter versus green fluorescence (531/40 nm) with blank (left), PE microspheres (middle) and ground PP samples (right), stained with NR at 10 µg/mL at room temperature for 30 mins. Blank and microspheres samples have been filtered between staining step and FC analysis, adapted from [49].

Fig. 4

particle concentration of MP (blue) and NP (green) in 9 simulated water samples measured by FC: 3 ultrapure water samples (S1–S3), 3 tap water samples (S4–S6), and 3 lake water samples (S7–S9). Adapted from [40] under the Creative Commons Attribution License (CC BY) [40].

Fig. 5

Schematic representation of the full quantification process of MNPs by FC.