Identification of microplastics using Nile Red (NR) Staining

Source: Sturm, M.T., Horn, H. & Schuhen, K. The potential of fluorescent dyes—a comparative study of Nile red and three derivatives for the detection of microplastics. Anal Bioanal Chem 413, 1059–1071 (2021). https://doi.org/10.1007/s00216-020-03066-w

The development of rapid and precise methods for identifying and quantifying microplastics in environmental monitoring studies is crucial. Nile Red (NR), a hydrophobic and photochemically stable dye, has been widely used for microplastic research. NR is soluble in nonpolar solvents and exhibits fluorescence in the visible spectrum, making it suitable for visualizing and quantifying hydrophobic molecules and polymers.

NR staining is a fluorescent technique used to detect microplastics in samples. The process involves applying NR dye to isolated microplastic particles and examining them under a fluorescence microscope. The resulting images are used for quantification and characterization of microplastics. Combining NR staining with spectroscopic analysis, such as FT-IR microscopy, can further enhance the identification of microplastics in samples.

Current methods for identifying microplastics face challenges when dealing with particles with the size below 200 μm and those covered by non-plastic solids. NR staining can help detect hidden microplastics in such cases, facilitating subsequent spectroscopic analysis. Installing a fluorescent filter on an FT-IR microscope allows for immediate confirmation of spectroscopic identification after fluorescence microscopy. This combined approach reduces the risk of missing microplastics in field samples and saves time compared to spectroscopic analysis alone.

Improvements in microplastic identification methods are necessary for effective monitoring using various sampling techniques and different particle sizes. NR staining, along with complementary spectroscopic analysis, offers a promising solution for the rapid detection and characterization of microplastics in environmental samples.

One limitation of the NR staining method is the co-staining of natural organic material, which can interfere with the identification of microplastics. It is necessary to remove natural lipids and organic matter from samples before staining. Additionally, some organic remnants may not be fully stained or may show a dim glow, making it difficult to distinguish them from microplastics. Highly weathered polymers and unknown newly synthesized polymers pose challenges in matching them with the FT-IR spectrum library.

Due to the presence of NR-stained unidentified particles and the variability of NR-stained organic remnants, it is not recommended to rely solely on the NR staining method for quantifying microplastics in field samples. Further improvements are needed to develop methods that can effectively remove non-plastic organic materials from environmental samples and ensure accurate quantification of microplastics.

Ref

1. W.J. Shim et al. (2016), “Identification and quantification of microplastics using Nile Red staining”, Marine Pollution Bulletin xxx 
2. Mariano et al., (2021), “ Micro and Nanoplastics Identification: Classic Methods and Innovative Detection Techniques”, Front. Toxicol. 3:636640. doi: 10.3389/ftox.2021.636640
3. V.C. Shruti et al., (2022), “Analyzing microplastics with Nile Red: Emerging trends, challenges, and prospects”, Journal of Hazardous Materials 423, 127171

By: Moe Thazin Shwe, SOLEN Research Associate – IPC panel member
Edited by: Hendra WINASTU, SOLEN Principal Associate – IPC panel coordinator
Date: 19 June 2023
Article#: SOLEN-IPC-0021