Dyeing to Know: Optimizing Solvents for Nile Red Fluorescence in Microplastics Analysis

With the escalation of microplastic (MP) pollution and the laborious nature of existing MP identification methods, new approaches for large-scale sampling of MPs in the environment are necessary. A promising solution lies in the fluorescence staining of Nile Red (NR), whose fluorescence is polarity-dependent, offering the potential for classification based on fluorescence. However, the choice of carrier solvents to dissolve NR remains unstandardized, and methods to represent and differentiate the fluorescent behavior of MPs are lacking. To address this gap, we conducted tests on eight NR-carrier solvents (n-hexane, chloroform, acetone, methanol, ethanol, acetone/hexane, acetone/ethanol, and acetone/water) applied to ten different types of MPs (HDPE, LDPE, PP, EPS, PS, PC, ABS, PVC, PET, and PA). We compared their fluorescence behavior (fluorescence intensity and Stokes shift), evaluated their effects on polymer degradation, and assessed the ability of different potential polarity measures and color spaces to accurately reflect Stokes shift for MP classification. Furthermore, Fenton oxidation was found to quench the fluorescence of natural organic matter (e.g., eggshells, fingernails, wood, and cotton), with minimal changes observed in NR-stained MPs. Our findings identified acetone/water [25%] as the best compromise, effectively mitigating the adverse effects of acetone while maintaining strong fluorescence behavior suitable for classification.