Quantification of the Uptake and Biodistribution of Nanoplastics in <i>Escherichia coli</i>

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 <i><i>Escherichia coli</i></i> (<i><i>E. coli</i></i>) 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 <i><i>E. coli</i></i> 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.