Fluorescent labeling of micro/nanoplastics for biological applications with a focus on "true-to-life" tracking

The increased environmental presence of micro-/nanoplastics (MNPLs) and the potential health risks associated with their exposure classify them as environmental pollutants with special environmental and health concerns. Consequently, there is an urgent need to investigate the potential risks associated with secondary MNPLs. In this context, using "true-to-life" MNPLs, resulting from the laboratory degradation of plastic goods, may be a sound approach. These non-commercial secondary MNPLs must be labeled to track their presence/journeys inside cells or organisms. Because the cell internalization of MNPLs is commonly analyzed using fluorescence techniques, the use of fluorescent dyes may be a sound method to label them. Five different compounds comprising two chemical dyes (Nile Red and Rhodamine-B), one optical brightener (Opticol), and two industrial dyes (Amarillo Luminoso and iDye PolyPink) were tested to determine their potential for such applications. Using commercial standards of polystyrene nanoplastics (PSNPLs) with an average size of 170 nm, different characteristics of the selected dyes such as the absence of impact on cell viability, specificity for plastic staining, no leaching, and lack of interference with other fluorochromes were analyzed. Based on the overall data obtained in the wide battery of assays performed, iDye PolyPink exhibited the most advantages, with respect to the other compounds, and was selected to effectively label "true-to-life" MNPLs. These advantages were confirmed using a proposed protocol, and labeling titanium-doped PETNPLs (obtained from the degradation of milk PET plastic bottles), as an example of "true-to-life" secondary NPLs. These results confirmed the usefulness of iDye PolyPink for labeling MNPLs and detecting cell internalization.