Development of a reliable preprocessing protocol for fluorescent micro- and nanoplastic analysis in human placental...

Concerns are arising about potential health risks of micro- and nanoplastics (MNPs) to human life, as their presence has been reported in human blood, stool, liver, lung tissue, breast milk, and placenta. However, little is known about particle numbers and morphology, which is essential information for developing reliable risk assessment. Therefore, such characterization of MNPs in human tissue is an important yet difficult task, which involves sample digestion as an essential step in the pretreatment of organic matrices. Successful digestion enables accurate characterization of MNPs using micro-spectroscopy. In this study, eight different enzymes or enzyme mixtures commonly used in digestion protocols were tested in four different buffers, to select the best combinations of enzymes and buffers for the preprocessing of human placental tissue for MNP (spectro-)microscopic analysis. Placenta tissue was spiked with fluorescent 200 nm, 500 nm, 1 μm, and 10 μm polystyrene (PS) MNPs to analyze morphological stability throughout the digestion and to determine recovery rates (RRs). For the optimal protocol, RRs of 98% ±6 (200 nm), 148% ±8 (500 nm), 147% ±8 (1 μm), and 81% ±8 (10 μm) were determined using confocal fluorescence microscopy (CFM). We explain values >100% by dye leaching and hypothesize that the leached dye can bind to organic residue from tissue with a similar size as the fluorescent PS particles, causing false positives when counting MNPs. Morphological changes were not observed for the final digestion protocol both with CFM and scanning electron microscopy. Hence, we demonstrate an optimized enzymatic digestion protocol to digest (placental) tissue and report on the accuracy of the characterization of model MNPs using micro-spectroscopy, which will enable further research with an emphasis on sub-micron (<1 µm) sized plastic particles.