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

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 (ωfinal = 10%) and NaCl (ωfinal = 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.