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Nile Red staining for nanoplastic quantification: Overcoming the challenge of false positive counts due to fluorescent aggregates
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Researchers refined a Nile Red dye-based method to count nanoplastic particles in water by reducing dye concentration to near-nanomolar levels, minimizing false positives from dye aggregates that mimic plastic particles. Using this approach they detected approximately 250 nanoplastics per nanoliter in water from plastic bottles and cartons — roughly ten times more than in tap water.
The true extent of plastic particle pollution is largely unknown, particularly for particles <1 µm (nanoplastics), as they are difficult to detect. Here, we expand the Nile Red (NR) staining approach often used to visualize microplastics to the quantification of nanoplastics. Using NR staining for nanoplastic quantification is largely unexplored due to the formation of fluorescent NR aggregates which cause false positive counts in single particle counting. Here, we study the number and size of the NR aggregates formed as a function of NR concentration and show that with decreasing NR concentration, the number and size of the NR aggregates drops. At nanomolar concentrations, the number of NR aggregates is low, while staining of nanoplastics at these concentrations still results in signals that are sufficiently bright for single particle detection. To challenge and verify our approach, we spiked a drinking water sample with known amounts of nanoplastics. After quantification of the total amount of fluorescent nanoparticles and considering NR aggregates and the added amounts of nanoplastics, we find a stable number of NR positive nanoparticles for the drinking water sample, thus verifying our approach. To demonstrate the direct applicability of our method, without any pre-analytical treatment, we determined the number of NR positive nanoparticles in different drinking water samples. In water obtained from plastic bottles and plastic-lined cartons, we detected approximately 250 nanoparticles/nL, which is well above the level of detection of our method. In tap water, we found approximately 10 times fewer NR positive nanoparticles. Our study demonstrates that staining at nM NR concentrations combined with a careful characterization of the number of NR aggregates in solution allows for using single-particle counting for nanoplastic quantification in water samples. We foresee that this approach can contribute to filling the gap in knowledge on the abundance of nanoplastics.
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Nile Red staining for nanoplastic quantification: Overcoming the challenge of false positive counts due to fluorescent aggregates
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Researchers expanded the Nile Red staining method to quantify nanoplastics smaller than 1 µm, identifying fluorescent aggregates as a source of false positive counts and developing methodological corrections to overcome this challenge and improve the accuracy of nanoplastic detection in environmental samples.
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Scientists developed an improved method for staining microplastics with a fluorescent dye (Nile red) that embeds the dye inside the plastic particles rather than just coating the surface, resulting in much brighter and more reliable detection. Combined with high-speed flow cytometry, the technique can rapidly count microplastic particles smaller than 10 µm in environmental water samples with recovery rates above 99%. Faster and more accurate counting methods like this are important for scaling up microplastic monitoring across many water sources.
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Scientists developed a better way to detect tiny plastic particles in bottled water by fine-tuning a special dye test, making it more accurate at spotting microplastics of different sizes. When they tested seven brands of bottled water (still and sparkling, in both plastic and glass bottles), they confirmed that a majority of the flagged particles were indeed plastic, a useful step toward more reliable methods for tracking how much plastic we might be drinking.
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