Comprehensive assessment of factors influencing Nile red staining: Eliciting solutions for efficient microplastics analysis

Highlights: • The H₂O₂ and NaCl addition lowered the fluorescence intensity of biogenic materials and reduced their interferences • The Nile red-stained microplastics were preliminarily categorized by merged fluorograph and combined fluorescent index • The glass transition temperature (T_g) is a crucial factor influencing the fluorescence intensity of microplastics • An elevated staining temperature promoted the fluorescence intensity of microplastics • A simplified and streamlined Nile red staining protocol was proposed Monitoring microplastics in the environment based on the Nile red staining protocol has proven to be a newly emerged method in several instances. However, the methodology is still having the limitations of susceptibility, indiscrimination, and complexity, etc. The objectives of this paper are to explore the effects of wavelength, temperature, H₂O₂ and NaCl addition, plastic property, and fluorescent index on the Nile red staining in microplastics analysis and propose solutions to these inadequacies. Sample co-stained with H₂O₂ (ω_final = 10%) and NaCl (ω_final = 8.8%) will lower the fluorescence intensity of biogenic materials and reduce their interferences. Based on the fluorescence color and intensity of fused fluorographs, the combined fluorescent index for twelve microplastics was significantly different, thus could be preliminarily distinguished. An elevated staining temperature is propitious to fluorescent tagging with Nile Red. Finally, an improved protocol was proposed, which made the methodology streamlined in microplastics analysis.