Label-free non-destructive spectroscopic detection of mixed microplastic uptake and differential effects on intestinal epithelial cells

Microplastics are an environmental and dietary contaminant of significant concern for human health due to chronic exposure and emerging evidence of cellular impact. However, understanding of this impact is limited by challenges in their detection and identification in biological samples. This study aimed to use Optical Photothermal Infrared Spectroscopy (O-PTIR) to detect and characterize real-world microplastics internalized by intestinal cells, and to investigate the cellular impact of such exposure. An intestinal epithelial cell line (IEC-6) was exposed to physically degraded fluorescent microplastics (25-100 μg/mL) made of four polymers for 24 h. O-PTIR reference spectra were acquired on single dyed primary plastics before fluorescent guided O-PTIR was used to identify and characterize microplastics in IEC-6 cells. We determined that the limit of resolution under these conditions was particles of ∼ 1 μm. Microplastics from all polymer types were successfully detected and discriminated using O-PTIR data alone. Importantly, principal component analysis of cytoplasmic O-PTIR spectra was used to identify microplastic-induced metabolic shifts. This chemometric analysis suggested that microplastic exposure resulted in oxidative stress and metabolic disruption, indicated by conformational protein changes-including early signs of misfolding and aggregation-which are hallmarks of a stress-adaptive cellular response. Our findings demonstrate that O-PTIR is an efficient, non-destructive technique for label-free microplastic studies in biological samples, enabling the identification, localization and characterization of heterogeneous microplastics, while simultaneously revealing their impact on cellular function and composition.