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Multimodal Analysis of Microplastics in Drinking Water using a Silicon Nanomembrane Analysis Pipeline
Published on: June 13, 2025
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Highly efficient Nile red staining for the rapid quantification of microplastic number concentrations using flow
Yuki Kuruma1,2, Hiromu Sakurai2, Tomoaki Okuda1
1Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa, 223-8522, Japan.
Analytical Methods : Advancing Methods and Applications
|November 13, 2025
Summary
This study introduces a new swelling-mediated Nile red staining method to improve microplastic detection. This technique enhances fluorescence for accurate quantification of small microplastics using flow cytometry.
Area of Science:
- Environmental Science
- Analytical Chemistry
- Materials Science
Background:
- Fluorescent staining with Nile red (NR) is crucial for quantifying microplastics (MPs) using optical methods like flow cytometry (FCM).
- Conventional NR staining often suffers from low fluorescence intensity due to variable polymer types and surface characteristics, limiting the detection of smaller MPs.
- This necessitates improved protocols for sensitive and accurate MP quantification.
Purpose of the Study:
- To develop and validate an improved swelling-mediated Nile red (NR) staining protocol for enhanced microplastic (MP) detection and quantification.
- To enable the identification and accurate counting of MPs, particularly those smaller than a few micrometers, using flow cytometry (FCM).
- To assess the efficiency and reliability of the new staining method in various MP sizes and simulated environmental matrices.
Main Methods:
- A novel swelling-mediated NR staining method was developed, encapsulating NR within MPs for stronger fluorescence compared to surface adsorption.
- The protocol was optimized using polystyrene microsphere suspensions (2, 5, and 10 µm) with known concentrations.
- Recovery tests were performed using simulated environmental samples (tap water matrix) and analyzed via flow cytometry (FCM).
Main Results:
- The swelling-mediated NR staining method significantly enhanced MP fluorescence intensity, improving detection sensitivity for MPs down to a few micrometers.
- Optimized protocols achieved near-unity staining efficiencies for polystyrene particles.
- Recovery rates in simulated samples were high, ranging from 0.846 to 0.993, with variations attributed to FCM counting efficiency rather than staining uncertainty.
Conclusions:
- The swelling-mediated NR staining technique offers a robust and sensitive method for quantifying microplastics, especially smaller particles.
- This approach, when coupled with high-throughput FCM, provides a rapid and reliable tool for analyzing microplastics in environmental samples.
- The method overcomes limitations of conventional staining, paving the way for more comprehensive MP monitoring.
