A novel method to detect nano-and microplastics in vivo

(Nanowerk Spotlight) Plastic debris is accumulating in oceans at a staggering rate. The quantity of plastics flowing into waterways and, ultimately, into the oceans is called marine plastic leakage. Estimates for its size vary from study to study, averaging about 12 million tonnes a year (read more: The Marine Plastic Footprint; pdf).
Microplastics (< 5 mm in size), may be the most abundant form of plastic pollution in the ocean – scientists estimate that the ocean contains 8.3 million pieces of plastic mini-microparticles (particles smaller than 333 µm) per cubic meter of water (source).
While plastic debris of any size is harmful to the marine flora and fauna, recent research suggests that the smallest of these plastics, nanoplastics, affect the composition and diversity of our intestinal microbiome and that this can cause damage to our health (Science Bulletin, "Insights into nanoplastics effects on human health").
"Microplastics can be detected either directly in environmental samples – i.e. water, soils, sediments, aerosols – or within the organisms harvested from a range of habitats, allowing for evaluation of biological impact along with qualitative and quantitative analysis of microplastics sizes, chemistry, surface coat and biodistribution," Professor Rawil Fakhrullin from the Kazan Federal University, Republic of Tatarstan, RF, tells Nanowerk. "A plethora of techniques is currently employed to identify microplastics based on thermal analysis, Fourier-transform infrared spectroscopy,reflectance micro-Fourier-transform (FT-IR) imaging, focal array FT-IR hyperspectral imaging, near infrared spectroscopy and refraction index analysis, Raman microspectroscopy, surface-enhanced Raman scattering microscopy and fluorescence microscopy."
However, as he points out, no previous study has reported experimental results demonstrating 1) resolving nanoplastics down to 100 nm, 2) visualizing micro- and nanoplastics in vivo, and 3) differentiating between microplastics of different chemical composition within the same biological specimen.
To address this challenge, Fakhrullin and his team have developed a novel optical spectroscopy-based methodology in the VIS-NIR range (400-1000 nm), capable for differentiating between chemically different micro- and nanoplastics confined within invertebrate intestines. The technology is based on dark-field microscopy, which is capable of visualizing particles smaller than the bright-field optical microscopy resolution limit.
They report their findings in Environmental Pollution ("Dark-field hyperspectral microscopy for label-free microplastics and nanoplastics detection and identification in vivo: a Caenorhabditis elegans study").
dark-field microscopy image of a mixture of colorless and pigmented (blue and red) polystyrene microspheres
a) Dark-field microscopy image of a mixture of colorless and pigmented (blue and red) 1 µm polystyrene microspheres in water; b) hyperspectral images merged with SAM algorithm-based mapping. (Image: Prof. Fakhrullin) (click on image to enlarge)
In this work, for the first time, polymer nanoparticles down to 100 nm were successfully visualized and chemically mapped in water suspensions and in vivo, employing a versatile Caenorhabditis elegans nematode as a model organism.
The researchers also succeeded in dark-field hyperspectral imaging of microsized polymethacrylate and melamine resin particles, which were also spatially mapped in nematodes using spectral libraries-based mapping algorithm.
"Our method doesn't require any specific labels or sample pre-treatment," Fakhrullin points out. "If compared to other spectroscopy-based methods, such as Raman microspectroscopy, dark-field hyperspectral microscopy is cheaper and easier to perform. In addition, this technology allows for the fast observation of relatively large specimens, such as whole microscopic animals, where the distribution of very few isolated particles can be located within minutes."
The team is confident that their technique will find applications in studies aimed at elucidation of nanoplastics and microplastics endocytosis pathways, biodistribution in tissues and organs, transgenerational studies and nanotoxicology.
distribution of polystyrene microparticles in C. elegans
a) Dark-field microscopy images and b) corresponding hyperspectral images merged with SAM algorithm-based maps demonstrating the distribution of 1 µm polystyrene in C. elegans. (Image: Prof. Fakhrullin) (click on image to enlarge)
The scientists' future work will be focused on adjusting the imaging technology to detect other types of nano- and microplastics. In their present study, they used polystyrene (both colorless and pigmented), which is among the most produced polymers worldwide and hence one of the greatest consequence to the environment.
They are also planning to investigate the detection limits of this technology, both in terms of particle size and particle concentrations. Microplastics are normally classified as any polymer with a size below 5 mm, however the most challenging is the detection of nanoscale plastics with sizes of 1 micron and less at least in one dimension.
"We expect that plastic particles with diameters down to 20 nm can be detected because similar non-plasmonic particles of comparable size, though chemically different, have already been visualized both in pure suspensions and in more complex media by us and others using dark-field hyperspectral microscopy," Fakhrullin concludes. "As for particle concentration, this is challenging because it is difficult to control the uptake of minute amounts of plastics by the model animals, therefore this part of our study will require an advanced protocol for particles delivery and reference concentration determination."
Michael Berger By – Michael is author of three books by the Royal Society of Chemistry:
Nano-Society: Pushing the Boundaries of Technology,
Nanotechnology: The Future is Tiny, and
Nanoengineering: The Skills and Tools Making Technology Invisible
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