A Robust Multistep Digestion Method for Microplastics Detection in Human Tissue by MicroRaman Analysis

The presence of microplastics (MPs) in human tissues has raised growing concerns, necessitating robust protocols for their reliable extraction and analysis. This study systematically evaluated and optimized digestion protocols to efficiently process a variety of human tissues-placenta, lung, kidney, adipose tissue, muscle, spleen, liver, thyroid, and brain-while preserving the integrity of MP particles. Initial assessments employing single-reagent protocols such as nitric acid (HNO₃), proteinase K enzymatic digestion, and Fenton oxidative digestion demonstrated limited effectiveness, due to incomplete tissue breakdown or formation of turbid digestates that hindered filtration. Building upon these results, combined digestion approaches were investigated to improve organic matter removal and facilitate filtration through fine pore-size filters (0.2 μm). The optimized 3-day protocol included an initial oxidative Fenton digestion followed by enzymatic digestion (proteinase K). The final step involved lipid removal through ethanol addition and sonication, resulting in clear digestates amenable to filtration. This protocol efficiently digested complex tissue matrices, reducing filter clogging at 1-μm size pore and preserving various common MP polymers, including low-density polyethylene (LDPE), polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), and polyamides (PA6 and PA12). Application of the optimized digestion allowed successful isolation and characterization of MPs using optical microscopy and Raman spectroscopy. The method showed improved reproducibility and reliability over single-reagent protocols, making it suitable for comprehensive MP analysis in human tissues. The application of an efficient and robust protocol for tissue digestion may contribute to advance human exposure assessment and toxicological studies related to MP contamination.