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A Robust Multistep Digestion Method for Microplastics Detection in Human Tissue by MicroRaman Analysis
Jennifer P Pascali1, Lucio Litti2, Arianna Fornasari1
1Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy.
Abstract:
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.
Insights
This study developed an optimized 3-day protocol for extracting microplastics (MPs) from diverse human tissues. The new method ensures MP integrity and clear digestates for reliable analysis, advancing human exposure assessments.
Area of Science:
- Environmental Science
- Toxicology
- Analytical Chemistry
Background:
- Microplastic (MP) contamination in human tissues is a growing concern.
- Reliable extraction and analysis of MPs from complex human tissues are challenging.
- Existing single-reagent digestion protocols are often ineffective, leading to incomplete tissue breakdown or filtration issues.
Purpose of the Study:
- To systematically evaluate and optimize digestion protocols for efficient MP extraction from various human tissues.
- To develop a robust method that preserves MP integrity and facilitates filtration.
- To enable accurate human exposure assessment and toxicological studies.
Main Methods:
- Evaluation of single-reagent protocols (nitric acid, proteinase K, Fenton digestion).
- Development and optimization of a combined 3-day digestion protocol involving Fenton digestion, enzymatic digestion (proteinase K), and lipid removal (ethanol/sonication).
- Application of optical microscopy and Raman spectroscopy for MP characterization.
Main Results:
- The optimized 3-day protocol effectively digested complex human tissues (placenta, lung, kidney, etc.), yielding clear digestates.
- The protocol reduced filter clogging and preserved common MP polymers (LDPE, PET, PTFE, PA6, PA12).
- The method demonstrated improved reproducibility and reliability compared to single-reagent protocols.
Conclusions:
- An efficient and robust 3-day digestion protocol was established for microplastic analysis in human tissues.
- This optimized method facilitates the isolation and characterization of microplastics, crucial for human health risk assessment.
- The protocol supports advancements in understanding human exposure and the toxicological impacts of microplastic contamination.
