Quantification of the Uptake and Biodistribution ofNanoplastics in Escherichia coli

Nanoplastics (NPs) are prevalent in the environment, posing risks to ecosystems and human health. While research into their effects on bacterial activity has increased, the mechanisms underlying NP-bacteria interactionsspecifically whether NPs penetrate cells or adhere to the cell surfaceremain poorly understood. This knowledge gap largely stems from the absence of quantitative analytical methods. Herein, we developed a novel approach combining lysozyme treatment with pyrolysis gas chromatography–mass spectrometry (Py-GC/MS) to differentiate between intracellular and cell wall-bound NPs in Escherichia coli (E. coli) quantitatively. The method involves selective removal of the bacterial cell wall using lysozyme, protein corona-induced extraction to enrich cell wall-bound NPs, and hydrogen peroxide digestion to eliminate protoplast interference before Py-GC/MS analysis. Validation with europium (Eu)-labeled NPs, quantified by inductively coupled plasma mass spectrometry (ICP-MS), confirmed the method’s accuracy and reliability. Using this approach, we found that after NP exposure, only a small fraction (9.6–10.5%) of NPs penetrated E. coli cells, while the majority (36.9–63.8%) adhered to the cell surface. Transmission electron microscopy further corroborated these findings. Consequently, this work provides a robust tool for the quantification of NP uptake and biodistribution in bacterial systems, advancing our understanding of NP–microorganism interactions and their environmental implications.