Nile Red staining for nanoplastic quantification: Overcoming the challenge of false positive counts due to fluorescent aggregates

The true extent of plastic particle pollution is largely unknown, particularly for particles <1 µm (na-noplastics), 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 drop. 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 ap-proach, we spiked a drinking water sample with known amounts of nanoplastics. After quantifica-tion of the total amount of NR positive particles and considering NR aggregates and the added amounts of nanoplastics, we find a stable number of NR positive particles for the drinking water sample, thus verifying our approach. To demonstrate the direct applicability of our method, with-out any pre-analytical treatment, we determined the number of NR positive particles in different drinking water samples. In water obtained from plastic bottles and plastic-lined cartons, we detect-ed approximately 250 particles/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.