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Dyeing to Know: Harmonizing Nile Red Staining Protocols for Microplastic Identification
Summary
Researchers systematically evaluated eight carrier solvents for Nile Red fluorescence staining to improve microplastic identification and classification. The study identified an acetone-water mixture as the optimal solvent, balancing strong fluorescence performance with minimal polymer degradation, and demonstrated that Fenton oxidation effectively eliminated false-positive fluorescence from natural organic materials.
The increasing prevalence of microplastic (MP) pollution and the labor-intensive nature of existing identification methods necessitate improved large-scale detection approaches. Nile Red (NR) fluorescence, which varies with polarity, offers a potential classification method, but standardization of carrier solvents and fluorescence differentiation techniques remains lacking. This study evaluated eight NR-carrier solvents (n-hexane, chloroform, acetone, methanol, ethanol, acetone/hexane, acetone/ethanol, and acetone/water) across ten common MP polymers (HDPE, LDPE, PP, EPS, PS, PC, ABS, PVC, PET, and PA). Fluorescence intensity, Stokes shift, and solvent-induced polymer degradation were analyzed. The study also assessed HSV (Hue/Saturation/Value) color spaces for Stokes shift representation and MP differentiation. Fenton oxidation effectively quenched fluorescence in natural organic matter (e.g., eggshells, fingernails, wood, cotton) while preserving NR-stained MPs. Acetone/water [25% (v/v)] emerged as the optimal solvent, balancing fluorescence performance and minimal degradation.