Development of a Nile Red staining method for microplastic identification and quantification

Abstract

Nile Red (NR), a fluorescent dye, which was well known to dye neutral lipid in cell and tissuesample. It is strongly fluorescent only in the presence of a hydrophobic environment. Applicabilityof NR for identification and quantification of microplastics was tested and stainingcondition was optimized in this study. Micro-sized polyethylene (PE), polypropylene (PP) andexpanded polystyrene (EPS) were used as model plastics. A number of carrying solvents weretested. NR demonstrated good staining capability in n-hexane without damage on both whiteand black polycarbonate (PC) filter papers. But, because low solubility of NR in n-hexane,stock solution was made in acetone and then diluted with n-hexane in working solution (5 and50 mg/L). Stained polymer particles were recognized better in green fluorescence (Ex. 534-558and Em. 515-565) than red (Ex. 534-558 and Em. > 590) without background (PC filterpaper) staining under a fluorescent microscope. Both the NR concentrations were enough todye three polymer particles, but 50 mg/L solution showed higher fluorescence than 5 mg/L solution.Fluorescence intensity was in the order of EPS > PP > PE. The developed NR stainingmethod was successfully applied to identify and quantify the fragmented polymer particles in alaboratory accelerated mechanical abrasion study. Staining polymer particles with NR is easyand fast to identify and quantify polymer pa of microplastics was tested and stainingcondition was optimized in this study. Micro-sized polyethylene (PE), polypropylene (PP) andexpanded polystyrene (EPS) were used as model plastics. A number of carrying solvents weretested. NR demonstrated good staining capability in n-hexane without damage on both whiteand black polycarbonate (PC) filter papers. But, because low solubility of NR in n-hexane,stock solution was made in acetone and then diluted with n-hexane in working solution (5 and50 mg/L). Stained polymer particles were recognized better in green fluorescence (Ex. 534-558and Em. 515-565) than red (Ex. 534-558 and Em. > 590) without background (PC filterpaper) staining under a fluorescent microscope. Both the NR concentrations were enough todye three polymer particles, but 50 mg/L solution showed higher fluorescence than 5 mg/L solution.Fluorescence intensity was in the order of EPS > PP > PE. The developed NR stainingmethod was successfully applied to identify and quantify the fragmented polymer particles in alaboratory accelerated mechanical abrasion study. Staining polymer particles with NR is easyand fast to identify and quantify polymer pa