A Single-Cell Multiparameter Cytometry Platform for Investigating Nanoplastics and Lead in Ferroptosis Regulation

Abstract Multidimensional single-cell analysis is indispensable for studying complex biological processes. Micro-/nanoplastics (MNPs) act as carriers of toxic metals. Studies have shown that a combination of the two can induce ferroptosis in cells. Reports on their combined toxic effects have described both antagonistic and synergistic interactions, most of which are based on traditional population-level assays. To further investigate this issue, we developed an integrated platform for three-parameter single-cell analysis (termed CytoLM Plus 2.0). It synchronously couples dual-channel laser-induced fluorescence (LIF) with inductively coupled plasma mass spectrometry (ICP-MS) and employs a K-means algorithm to precisely align the multiparameter signals from individual cells. Specifically, the limit of detection for sodium fluorescein with a 473 nm laser is 0.65 pmol/L, while that for Cy5 with a 635 nm laser is 7.9 pmol/L. CytoLM Plus 2.0 was applied to study ferroptosis induced by 80 nm polystyrene nanoplastics (PSNPs), lead (Pb), or their combination. Cell population analysis indicates that PSNPs reduce lead bioavailability, producing an antagonistic effect. However, single-cell analysis reveals pronounced synergistic effects in certain subpopulations, and this intercellular heterogeneity may be closely related to the cell cycle. This study demonstrates that the interaction between MNPs and metals is not uniform but rather a context-dependent spectrum shaped by single-cell physiology. The present work establishes a multimodal quantitative framework for single-cell phenotypes, elemental loading, and heterogeneous responses. It demonstrates the feasibility of integrating optical flow cytometry with mass cytometry, providing a novel strategy for investigating complex biological processes.