“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.”
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Reporting in the Journal of Hazardous Materials, researchers at the University of Notre Dame Australia, Curtin University, and Monash University addressed a critical challenge in microplastic research: understanding microplastic impact is limited by challenges in their detection and identification in biological samples. Commonly accepted methods for detecting microplastics include pyrolysis gas chromatography, FTIR spectroscopy, and Raman spectroscopy, each with their own limitations, such as destruction of samples, poor resolution or interference by fluorescence respectively. To address this gap, the research team employed Optical Photothermal Infrared Spectroscopy (O-PTIR) to detect and characterize real-world microplastics internalized by intestinal cells. The work used irregular real-world microplastics made from household items composed of PET, PTFE, PS and PVC.
An intestinal epithelial cell line (IEC-6) was exposed to physically degraded fluorescent microplastics at concentrations of 25-100 μg/mL made of four polymers for 24 hours. Confocal microscopy showed that fluorescent microplastics of varying shapes and sizes were internalized and adhered to IEC-6 cell monolayers. 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. The limit of resolution under these conditions was determined to be particles of approximately 1 μm. Microplastics from all polymer types were successfully detected and discriminated using O-PTIR data alone. Overlapping PET and PTFE microplastics produced distinct spectra, which were consistent with reference polymer spectra despite the proximity of these particles. The spectra acquired for microplastics still contained a substantial contribution of amide bands, yet the microplastic detection and polymer discrimination by O-PTIR was not confounded by embedment in tissue.
Principal component analysis (PCA) 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. Microplastic-exposed cells displayed reduced relative intensity in bands at 1684, 1670, and 1638 cm⁻¹ and a marked increase at 1656 cm⁻¹ and 1622 cm⁻¹. The enhancement at 1656 cm⁻¹ may reflect upregulation or stabilization of stress-associated proteins, while the emergence of the 1622 cm⁻¹ band suggests increased aggregated β-sheet content, commonly linked to protein misfolding and early aggregation events. Microplastic-exposed cells showed increased absorption at 1738 and 1706 cm⁻¹, which are indicators of lipid peroxidation and accumulation of oxidized lipid species. Simultaneous suppression of bands at 1448 cm⁻¹, 1248 cm⁻¹, and 1106/1062 cm⁻¹ suggests degradation or structural modification of membrane phospholipids, nucleic acid backbones, and glycoproteins.
The limitations of traditional techniques are largely overcome by the emerging availability of optical photothermal infrared (O-PTIR) spectroscopy, which is a non-contact, non-destructive, label free technique that achieves a high resolution and is not affected by fluorescence. This study used O-PTIR to identify and discriminate between microplastics of four different polymers within biological tissue. The spatial orientation of microplastics was observable, and microplastic fluorescence proved to be helpful but not necessary for microplastic identification, endorsing the label-free capabilities of O-PTIR. The results demonstrate that O-PTIR could be used to reveal tissue dysfunction local to microplastic deposition in clinical samples. The findings show that the optical capabilities, resolution and sensitivity of O-PTIR make it more advantageous for label-free non-destructive microplastic detection compared to traditional techniques. The researchers position O-PTIR as a promising platform for both mechanistic insights and potential clinical translation.
Authors: Charlotte E. Sofield, Edward Attenborough, Ryan S. Anderton, Anastazja M. Gorecki, Li Shan Chiu, Chidozie C. Anyaegbu, Kamila Kochan
First Author Institution: Charlotte E. Sofield, University of Notre Dame Australia and Curtin University
