Single Nanoplastics Detection and Nano-Chemical Analysis of Surface Degradation in Commercial Bottled Water

Single Nanoplastics Detection and Nano-Chemical Analysis of Surface Degradation in Commercial Bottled Water | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Single Nanoplastics Detection and Nano-Chemical Analysis of Surface Degradation in Commercial Bottled Water Francesco Ruggeri, Clementina Vitali, Michel M.W. Nielen, Hans-Gerd Janssen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6719488/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Nanoplastics (NPs) are emerging as widespread environmental and food contaminants, posing significant concerns because of their potential to penetrate biological membranes, accumulate in tissues, and induce toxic effects. For understanding the impact of NPs on human health, a detailed characterization of their complex physicochemical properties is required. Yet, current analytical methods either lack single-particle spatial resolution, sensitivity, or specificity to detect and analyse NPs contaminating real samples. Here, for the first time, we detect and perform a nano-analytical characterisation of single NPs isolated from commercial drinking water as small as 30 nm; combining spatially confined hyper-concentration, fluorescence-guided nano-imaging, and chemical analysis via infrared nanospectroscopy. Our novel approach offers high-throughput single-particle analysis in real samples, enabling a multimodal characterisation of their 3D morphology, size, chemical identity, and surface degradation. This work paves the way to detect and analyse NPs in complex food matrices and biological systems, to study their interactions, fate, and toxicity. Physical sciences/Chemistry/Analytical chemistry/Infrared spectroscopy Earth and environmental sciences/Environmental sciences/Environmental impact Earth and environmental sciences/Environmental sciences/Environmental chemistry Physical sciences/Nanoscience and technology/Techniques and instrumentation/Imaging techniques Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Nanoplastics (NPs) – plastic particles smaller than 1 mm 1 – are emerging contaminants generated by the degradation of larger plastics debris 2 , including microplastics (MPs) measuring less than 5 mm 1 . As MPs have become widespread environmental contaminants 3,4 , NPs are expected to be present wherever MPs are found 2 . However, data on NP occurrence remains scarce due to the analytical challenges associated with their detection and characterization in real samples. The detection of MPs in food 5 has increased awareness of human exposure 6 . The potential concurrent presence of NPs is of particular concern because their sub-micrometer size and increased surface-to-volume ratio may facilitate their penetration across biological membranes, including the blood-brain barrier 7 . Studies involving rodent models have shown that NPs ingested through spiked water or feed can reach various organs, especially the brain 8,9 . Recently, MPs and NPs have also been detected in human brains, with increased concentration in decedents with diagnosed dementia 10 . Increasing evidences of NP neurotoxicity link them to oxidative stress, inhibition of acetylcholinesterase activity, cerebral thrombosis, and neurological dysfunction 11,12 . Compared to MPs, NPs exhibit increased capacity to accumulate in brain tissues, disrupt protein folding, and initiate the aggregation of amyloid proteins, a key molecular process in the onset of neurodegenerative diseases such as Alzheimer’s and Parkinson’s 13 . The analysis of NPs poses significant challenges, primarily due to their sub-diffraction limit size and higher surface reactivity, complicating both their isolation and their chemical identification 2,14 . The sub-micrometer size of NPs, coupled with the dominance of Brownian motion over sedimentation, limits the effectiveness of enrichment methods like dead-end filtration and density separation, commonly used for MPs 15,16 . The development of dedicated sample preparation procedures is complicated by the lack of environmentally representative reference materials 2,14 . Engineered nanobeads, the only commercial NP analytical standards, do not reflect the heterogeneity of true-life NPs originating from diverse fragmentation pathways 2,16 . As a result, approaches that are developed relying on uniform synthetic particles often prove ineffective for the analysis of NPs in real samples 16 . In principle, several analytical techniques have the sensitivity to detect NPs 15 . Nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) allow measuring NPs size distribution and concentration in liquid, but provide only bulk population data. NP analysis at single particle level can be achieved with electron microscopy (EM) and atomic force microscopy (AFM), which respectively yield 2-D and 3-D imaging with sub-nanometre resolution. These techniques yet lack chemical specificity, thus have been primarily applied to monitor NPs released during the controlled degradation of plastic items 17–24 . Pyrolysis gas chromatography mass spectrometry (Py-GC/MS) offers chemical identification of polymers based on their thermal degradation products, still providing only bulk mass-based quantitative results of the overall MP and NP contamination 25,26 . Nano-resolved vibrational spectroscopy techniques, such as hyperspectral stimulated Raman scattering (SRS) 27 and photothermal infrared spectroscopy (OPTIR) 28,29 , allow detecting individual NPs in the 200-500 nm range. Yet, the imaging capabilities of these methods rely on chemical mapping, hampering quantitative morphological characterization. Real NPs exhibit considerable heterogeneity in morphology and surface properties, including weathering and chemical degradation, relevant to their toxicity and fate in the human body 30,31 . Yet, such complexity cannot be resolved by any of the above techniques. AFM-based infrared nanospectroscopy (AFM-IR), combining the nanoscale spatial resolution of AFM with the chemical identification capabilities of vibrational spectroscopy, allows the quantitative assessment of 3-D morphology and chemical properties of single-molecules and polymers at the nano-scale 32–35 . While AFM-IR provides a highly promising solution for a comprehensive multidimensional analysis of NPs, only few preliminary studies have applied this technology to detect polymer-specific IR signatures of NPs isolated from real environmental samples 36,37 . Leveraging the full potential of AFM-IR for the analysis of NPs remains limited by practical constraints, arising primarily from the challenges associated with sample preparation – a fundamental step – as matrix interference and surface degradation complicate the analysis of nano-sized particles with an inherent low signal-to-noise ratio, especially when measuring on non-metallic substrates 38 . Due to the time-intensive nature of nanospectroscopy techniques, maximizing analyte concentration within a confined spatial area is critical for efficient measurements. Yet, this is particularly difficult for NPs, as conventional enrichment strategies are not readily transferrable to the nanoscale. Here, we develop a framework for the single particle physicochemical characterization in commercial bottled water of NPs as small as 30 nm. To enhance detection efficiency, we leverage spatially confined hyper-concentration on hydrophobic surfaces and perform fluorescence-guided nano-imaging and -chemical analysis using our recently developed single-molecule AFM-IR 39 ( Fig. 1 ). We first demonstrate our approach on standard polystyrene (PS) nano-beads, and polyethylene (PE) and poly(ethylene terephthalate) (PET) powders, spanning micro- to nano- scale. We then report the multimodal and correlative analysis of NPs isolated from real samples of bottled water, to unravel for the first time their physicochemical properties revealing significant surface degradation, which may critically influence their behaviour and potential toxicity. These achievements pose the basis for the development of accurate NPs standards and advancing our understanding of their interaction with and within biological systems. Results Statistical identification of NPs via fluorescence-guided nano-imaging Before starting the nano-analytical investigation of NPs in spiked and real water samples, we developed a fluorescence-guided nano-imaging method to prove the robust statistical discrimination of NPs against residual impurities naturally present on surfaces ( Fig. 2 ). Nano-imaging methods, such as AFM, require indeed the deposition of the sample on adequate substrates, and their sub-diffraction limit resolution allows to reveal the presence of residual impurities on the substrate, which are comparable in size to NPs, hence potentially interfering with their analysis. As a model system, we considered the nano-imaging analysis of PS nano-beads deposited on hydrophobic and IR-transparent ZnSe substrates. We investigated these substrates before and after the deposition of a mix of MP and NP analytical standards, to then allow fluorescence-guided analysis from the micro- to the nano-scale ( Fig. 2A ). Bare ZnSe substrates showed absence of fluorescent particles ( Fig. 2B ) and negligible autofluorescence ( Fig. 2C ) , proving to be suitable for the observation of Nile Red stained MPs. We then observed the behaviour of MPs/NPs deposited on the substrate in water suspension. Upon evaporation of the liquid phase, the PS beads concentrated in a spatially confined area with diameter <1 mm ( Fig. 2B, SI Video 1 ), decreasing the dimension of the region of interest for high-throughput nano-imaging analysis. The deposition of PS beads on the substrates leads to a significantly increased fluorescence signal ( Fig. 2C ). We then performed AFM mapping of ZnSe substrates before and after MPs/NPs deposition. The bare ZnSe substrate showed presence of residual nanoscale impurities ( Fig. 2D ). We statistically characterized their size by collecting AFM maps of randomly selected areas (10 × 10 µm, n = 9) on independent ZnSe substrates (n = 3). The residual particles had a height of 20 ± 10 nm and cross-sectional diameters (convoluted) of 50 ± 30 nm ( Fig. 2E ). Compared to other hydrophobic and IR-transparent surfaces, such as ZnS, ZnSe showed lower roughness (1.8 ± 0.4 nm) and a narrower size distribution of residual impurities ( Fig. S1-2 , Supplementary Notes 1-2 ). At the nano-scale, the impurities on the surface were easily recognizable, which facilitates their discrimination from the NPs ( Fig. 2D, E ), which represents a key advantage for improving the efficiency of time-intensive nano-scale analysis. To evaluate the statistical relevance of the discrimination of the NPs over the impurities, we plotted the height and diameter of the PS NPs (n = 88) over a 3D kernel density of the size of the impurities (n = 1452, Fig. 2F ), which outlines a 95% confidence interval neatly praising apart NPs from the impurities on the surface. These results proved that fluorescence-guided nano-imaging, although chemically blind, could be used to detect NPs apart from natural residual impurities already present on the surface with relevant statistical power. Standard PS NPs in Spiked Water Nano-imaging identified the PS NPs on the surface of analysis but was not able to provide chemical recognition. We thus next deposited on a ZnSe substrate a water suspension of PS nano-beads standards with 100 nm and 500 nm size to prove single-particle chemical analysis via AFM-IR ( Fig. 3A ). We preliminary performed a conventional bulk Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR) analysis of the PS standard to assign the major IR peaks of the material ( Fig. 3B , Fig. S3 , Table S1-7 , SI Notes 3 ). In the spectra, we observed the typical IR absorption bands of PS – 1600, 1582, 1492, and 1452 cm -1 – arising from the C-C stretching vibrations in conjugated six-membered rings 40 . We then characterized the PS standard by AFM-IR, which allowed to acquire the 3-D morphology and nano-localized infrared spectra of single nanobeads, of the two standards with size of 500 nm ( Fig. 3C ) and 100 nm ( Fig. 3D ). The AFM-IR analysis showed 3-D morphology of the nanobeads in excellent agreement with information provided by the producer of the standard, and the spectra showed the typical C-C stretching of PS, and with ATR-FTIR. Thus, AFM-IR proved capable of correlating the physicochemical properties of standard PS nanobeads – such as their size, shape, and chemical identity – in spiked water. Nano-Chemical Identification of PE and PET Powder MPs/NPs in Spiked Water We next applied AFM-IR for the analysis of irregular powder of PE and PET and spiked in water ( Fig. 4 ). These standard materials present variable shape, size, and surface properties, thus they more closely resemble MPs/NPs typically isolated from food or environmental matrices 16 compared to PS micro- and nano-beads. Of note, while our work is focused on NPs analysis, AFM-IR allows also high throughput characterisation of MPs. Suspensions of PE and PET MPs/NPs were deposited on ZnSe substrates ( Fig. 4A ). Before nano-chemical analysis, the bulk spectra of PE and PET MPs/NPs spiked in water were measured by ATR-FTIR ( Fig. 4B-C , Fig. S3 , Table S1-7 ). In the spectrum of PET ( Fig. 4B ), the peak at 1712 cm -1 was assigned to the carbon-oxygen (C=O) stretching vibration in the aryl carbonyl complex; the 1613 cm -1 , 1579 cm -1 , 1504 cm -1 , and 1406 cm -1 peaks to carbon-carbon (C-C) stretching vibration in the benzene ring; the peak at 1341 cm -1 to the twisting vibration of the methylene-oxygen bond (O-CH 2 ); the peak at 1241 cm -1 to the asymmetric vibration of the ester group (C-O-C). In the spectrum of PE MPs/NPs ( Fig. 4C ) a major peak at 1470 cm -1 was assigned to methylene (CH 2 ) scissor vibration; weaker bands at 1437 cm -1 and 1368 cm -1 were assigned to the methyl (CH 3 ) asymmetric and symmetric deformation, respectively; and the peak at 1304 cm -1 to the twisting vibration along the methylene (CH 2 ) chain. We then pursued the sing-particle nano-chemical characterisation of PET and PE MPs/NPs by AFM-IR ( Fig. 4D-F ). AFM-IR allowed to correlate the 3-D morphology of the particles via nano-imaging with their chemical identity and molecular composition via nano-localised IR spectra and chemical imaging ( Fig. 4D-F ). PET NPs showed irregular shapes and a high degree of similarity of the chemical spectra with those obtained by ATR-FTIR, with major peaks of absorption related to the C=O, C=C and C-O-C molecular bonds ( Fig. 4D ). This single particle multimodal morphological and chemical analysis was similarly possible for PE MPs/NPs, which showed pyramidal shape in the micro- and nano-size range, with a major IR peak associated to CH 2 absorption ( Fig. 4 E ). AFM-IR also allowed to correlate the morphology maps with chemical IR absorption maps of the NPs with high-resolution and sensitivity ( Fig. 4F ); IR maps at 1470 cm -1 and 1770 cm -1 – where PE is expected to show intense and null absorption, respectively – proved that the chemical signal arise purely from a single NP ( Fig. 4F ). Single-Particle Nano-polarimetry to detect MPs/NPs surface degradation In the AFM-IR spectra of the PE MP and the PE NP ( Fig. S4 and Fig. 4E ), while both particles are dominated by the methylene absorption at 1470 cm -1 , two additional broad bands with maxima of intensity at 1600 cm -1 and 1374 cm -1 are visible. These bands do not typically belong to the spectrum of pure PE, thus their identification required further considerations. As declared by the supplier, the studied PE material underwent a fluoro-oxidation during the manufacturing process, to introduce acid groups on the particle surfaces. Carboxylate salts typically exhibit absorption in the regions of 1620-1560 cm⁻¹ and 1420-1340 cm⁻¹, corresponding to the asymmetric and symmetric stretching of the carboxylate group (–COOH), respectively 40 . These additional bands could therefore be linked to the surface modification of the MPs/NPs. To substantiate the hypothesis of surface modification of our PE MPs/NPs, we exploited the nano-polarimetry capabilities of AFM-IR. Due to the electromagnetic enhancement of the optical field intensity at the apex of our gold-coated AFM tip, measurements performed irradiating the sample with p-polarized (90°) IR light result in spectra where the contribution of superficial layers is more significant 41 ; instead, spectra acquired with s-polarized IR light (0°), without enhancement at the tip, are more sensitive to the bulk chemical properties of the particles. We thus acquired single-particle spectra of PE MPs and NPs as a function of the polarization of the IR light. The spectra showed that the intensity of absorption of the band ascribed to the surface modification at 1600 cm -1 is stronger in the spectra acquired with surface sensitive (90°) polarization, and weaker in the spectra acquired with bulk sensitive (0°) polarization for both PE particles ( Fig. 4E, Fig. S4 ). The increased presence of carboxylate compared to methylene groups on the particle surfaces confirms that the unexpected absorption band at 1600 cm -1 and 1374 cm -1 in the IR spectra arises from surface modification, in agreement with what declared by the manufacturer. Overall, we could demonstrate that not only AFM-IR is able to chemically identify standard of spherical and irregular shape in water, spanning from the MP to the NP single particle scale, but also that this technique empowers the characterization of the surface properties of the particles, allowing the study of MP and NP weathering and chemical degradation. Spatially Confined Hyper-Concentration of MPs/NPs from real sample of bottled water The investigation of NPs contamination in real samples of commercial bottled water presents further challenges compared to the analysis of sample spiked with standard nanobeads, as discussed here and in previous studies 2,16 . The estimated concentration of MPs/NPs in bottled water of 2.4 ± 1.3 particle/L 27 is far lower than the optimal level for single-particle nano-scale analysis. Thus, hyper-concentration methods are required not only to concentrate the MPs/NPs in solution, but also to confine them in a surface of analysis that is compatible with micro-to-nano spatially resolved techniques and allows time-efficient analysis. To achieve spatially-confined hyper-concentration, we first pre-concentrated the samples, reducing the volume while retaining MPs/NPs, and then deposited them in order to dry within a spatially-confined surface with diameter <1 mm ( Fig. 5A ). We achieved 50x concentrated samples using a rotary evaporator. This operation resulted as well in the concentration of the minerals naturally present in bottled water, which added further complexity to the samples once deposited. To prevent the matrix from masking or interfering with the detection of plastic particles, we performed an additional rinsing step aimed at removing excess salts. An increasingly invasive handing of real water samples comes with a higher chance of procedural contamination, thus we performed the procedure on: analytical grade pure water ( Fig 5B ), analytical grade pure water concentrated by 50x ( Fig 5C ); bottled water ( Fig 5D ), and bottled water concentrated by 50x ( Fig 5E ). Fig. 5B-E show comparative brightfield and fluorescence images, via Nile Red staining, of the resulting spatially confined and hyper-concentrated samples, revealing the distribution of MPs/NPs by increased fluorescence intensity. A quantification of total fluorescence of each sample ( Fig. 5F ) showed that negligible MPs/NPs content is present in pure water, while 50x pre-concentration allowed the enhanced detection of MPs/NPs in bottled water. Thus, the presented hyper-concentration method overall allowed to spatially confine MPs/NPs on a finite area for nano-chemical analysis while maintaining procedural contamination at a negligible level. Physicochemical Identification and Degradation Analysis of NPs in real samples of bottled water We finally proved our framework combining spatially-confined hyper-concentration of particles suspended in water samples and fluorescence-guided nano-chemical imaging and spectroscopy able to detect and study NPs in real samples of commercial bottled water ( Fig. 6 and Fig. S6 ). Two bottled water samples were purchased from a local store. One was packaged in conventional PET, while the other was bottled in bio-HDPE, a bio-based high-density polyethylene derived from renewable resources such as sugarcane. After concentration by a factor of 50 and Nile Red staining, the samples were deposited onto ZnSe windows ( Fig. 6A ). We first performed our analysis on the water sample stored in a PET bottle. Fluorescence microscopy was used to identify MP/NP-rich areas within or around the deposited sample for subsequent nano-analysis ( Fig. 6B ). Despite NP analysis being the focus of the method developed in this study; the method is also suitable for MP analysis. Accordingly, we pointed the probe of the AFM-IR on a selected area of interest where MPs were localised ( Fig. S6 ). To identify the chemical identity of these MPs, we acquired nano-localised IR spectra, which showed two major bands relative to C=O and C-O-C molecular vibrations, aligning with the spectral signature of PET ( Fig. 6C ). We further performed nano-imaging to investigate the presence of NPs around the MPs, revealing numerous nanoparticles. We thus acquired nano-localised single-particle spectra to assess their chemical identify, on NPs as small as 30 nm in height ( Fig. 6D , Fig. S6 ). The spectra showed typical absorption bands of C=O, C=C, and C-O-C stretching vibrations, thus allowing to identify them as PET, in agreement with bulk FTIR ( Fig. S3 ). The spectra of PET MPs also showed additional absorption bands, which could be associated to the photo-oxidation and degradation of PET, likely resulting from sunlight exposure during transport and storage of water bottles. Exposure to UV light provides enough energy to break chemical bonds. Oxygen, then, can react with these broken bonds to form new groups such as quinones and aldehydes. Indeed, the observed peaks corresponded to IR absorption of quinones 42 (C=O stretching absorption, 1690-1655 cm -1 ) 40 and aldehydes 42 (CH rocking vibration, 1415-1350 cm -1 ) 40 . The spectra of PET NPs also showed signs of surface degradation ( Fig. 6D ). Compared to the spectra acquired by irradiating the samples with s-polarized IR light, the spectra obtained with p-polarized light present a decreased absorption intensity in correspondence to the ester COC asymmetric stretching vibration peak, which has been reported as a typical sign of PET photo-oxidation 42 . Furthermore, the shifts in the C=O to COC ratio suggest varying degrees of ester bond cleavage, indicating chain scission as a likely degradation pathway contributing to the formation of progressively smaller plastic particles. We next performed our analysis on water bottled in bio-HDPE, which also allowed to identify and characterise NPs physicochemical properties ( Fig. 6E-G ). The bulk IR signature of Bio-HDPE is dominated by CH 2 and CH 3 molecular vibrations ( Fig. 6E ). We further performed AFM-IR analysis, and the nanoscale morphology maps of a NP and a fibre isolated from bottled water packaged in bio-HDPE are reported, along with their spectra ( Fig. 6G , Fig. S6 ). The strong CH 2 scissoring absorption band and the weaker but visible symmetric deformation of methyl groups allowed to chemically identify the particles as PE. In the spectra of the particles isolated from the bio-HDPE bottle, two additional peaks at 1573 and 1422 cm -1 were observed, which may be assigned to the asymmetric and symmetric stretch of acetate ions (C=O) 40 . These chemical groups are typically associated with PE oxidation 43 , suggesting that PE NPs in water undergo significant degradation. We could thus, for the first time, demonstrate the single-particle multidimensional physicochemical characterisation of NPs in commercial bottled water, including the analysis of chemical modification involving either the whole particle or just its surface. These findings provide new insights into plastic degradation mechanisms at the nanoscale and may serve as a foundation for future investigations into the surface chemistry of environmental NPs, supporting the development of more environmentally relevant NP standards. Discussion We developed a framework combining spatially confined hyper-concentration of water samples with fluorescence-guided nano-imaging and -chemical analysis, enabling the detection and characterisation of NP at the single-particle level down to 30 nm size. Our approach overcomes the challenges posed by the sub-micrometre size of NPs and their typically low concentration in real samples. The enrichment of the sample by evaporation of the liquid phase, enabled the concentration of NPs across the entire size range, in contrast to conventional filtration-based methods, which fail to isolate particles smaller than the filter pore size 44 . The use of Nile Red as an indicator of MP/NP-rich areas guided the localization of the NPs on the surface, enhancing the analytical throughput. While previous attempt to detect NPs in dinking water 45 and biota 46 have cited the low concentration of analytes and the time-consuming nature of nano-chemical methods as obstacles to successful NP analysis, we demonstrated that with optimized sample preparation and methodology, the full potential of AFM-IR for comprehensive NP characterization can be realized. Our AFM-IR based approach enabled, for the first time, the simultaneous acquisition of nano-resolved 3D morphology and chemical properties of NPs in the biologically relevant size range of 10 nm to 1000 nm, following their isolation from both standard materials and real bottled water samples. Detailed morphological data are particularly significant, as the size and shape of NPs play a critical role in their transport, fate, and interaction with biological systems. Insights from drug design, which can be extended to NP research, have shown that particle size and shape determines cellular uptake efficiency and circulation time, with particles 200 nm being cleared from the bloodstream and accumulated in the liver and spleen 47 . Similarly, shape affects cellular uptake and tissue penetration 48 . Furthermore, surface chemistry of nanoparticles plays a key role in their interactions with biological systems 47,48 , affecting particle reactivity, ability to cross biological membranes, and ultimately the potential induction of adverse biological responses. The use of nano-polarimetry allowed us to investigate the surface chemistry of MPs and NPs. To the best of our knowledge, this is the first report detailing the surface modification of NPs isolated from real samples, providing a foundation for future research into degradation mechanisms at the nanoscale. Such insights will promote the development of more environmentally relevant NP standards, which will, in turn, support advancements across multiple areas of NP research. In conclusion, our approach represents a powerful platform for the comprehensive physicochemical characterization of NPs. By unlocking the full capabilities of AFM-IR, it paves the way for advancing our understanding of the environmental and health impacts of NP contamination, including the elucidation of the interactions between NPs and biological systems. Future investigations could leverage AFM-IR to explore critical questions regarding the role of ingested NPs in initiating biological responses, such as protein misfolding and aggregation, which have been implicated in the onset and progression of neurodegenerative diseases. Methods Chemicals and standards. Ultra-high molecular weight, surface-modified, polyethylene (PE) powder, with nominal size of 40-48 µm, was purchased form Sigma-Aldrich (Schnelldorf, Germany). Poly(ethylene terephthalate) (PET) powder and Poly(hydroxy butyrate)/poly(hydroxy valerate) 2% biopolymer (PHB), both with a nominal size of 300 µm, were purchased from Goodfellow (Hamburg, Germany). Polypropylene (PP) rod (Goodfellows) and poly(lactic acid) filament (3D Jake, Austria) were grinded in-house down to the microscale with a metal file. Polystyrene (PS) analytical standards with certified particle sizes (100 nm, 500 nm, 1 µm, 2 µm, 5 µm) were purchased from Sigma-Aldrich. Ultrapure water was obtained in-house from a MilliQ Water Purification System (Merk, Darmstadt, Germany).LC-MSgrade water was purchased from Rathburn (Walkerburn, United Kingdom), HPLC-grade acetone from Actu-All Chemicals (Oss, Netherlands), and HPLC-grade ethanol from (Supelco, Darmstadt, Germany). Nile Red crystals were purchased form Sigma-Aldrich. Tween20 was purchased form Sigma Aldrich. Real samples. Bottled water samples were purchased at a local store, including 500 mL PET bottle containing still mineral spring water; and 500 mL Bio-HDPE bottle containing still tap water. The entire content of each bottle was concentrated by a factor of 50 with a rotary evaporator operated at 40 °C and 45 mbar for ~8 hrs. Contamination prevention. To prevent environmental contamination of samples and standards, 100% cotton lab coats and particle-free nitrile gloves were worn during operations. In addition to the routine cleaning procedure, each piece of glassware was washed with ultrapure water and Tween20, rinsed with analytical grade acetone, and baked overnight at 550 °C. Fluorescence microscopy. Brightfield and fluorescence images were captured using an Olympus BX51 microscope, fitted with 4x, 10x, 20x, and 40x Olympus objectives, an Olympus SC50 RGB camera, an Olympus U-RFL-T UV lamp, and a bandpass filter with excitation wavelengths of 460-490 nm and emission wavelengths greater than 515 nm. The images were collected by built-in software cellSens; fluorescence images were processed in ImageJ (version 1.54) by subtracting the background (rolling ball radius of 25 px) and applying a median filer (radius of 5 px). For fluorescence observation, MPs/NPs were suspended in MilliQ water, tagged with Nile Red 49,50 , and deposited on ZnSe substrates. Protocol for total fluorescence analysis is reported in Supplementary Notes 1. Atomic force microscopy. AFM maps were acquired on a MultiMode 8 AFM and its built-in software Nanoscope (version 9.7, Bruker) operating in Peak Force tapping mode and equipped with ScanAsyst-air silicon tip with a nominal radius of ~2 nm and an elastic constant of about 0.4 N m -1 . Suitability for NPs analysis was assessed for ZnSe and ZnS substrates (Crystran Limited, Poole, United Kingdom) by characterization of the bare surface and AFM imaging of standard PS NPs. For the latter, 250 ppm suspensions of PS NPs were prepared in MilliQ water. Image flattening, root mean square roughness measurements, and particle analysis were performed in MountainsSPIP (version 10, Digital Surf, France) software. Protocols and results are reported in Supplementary Notes 2. Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy. ATR-FTIR analysis was performed using a Bruker Tensor27 spectrometer equipped with a diamond ATR element. Spectra were collected by built-in software OPUS (version 7.8, Bruker, USA). MP suspensions at a concentration of 10% (w/w) were prepared in ultra-pure water. Following a 30 s vortex agitation, a 0.5 μL aliquot was sampled and deposited on the ATR crystal. Each suspension was analysed in triplicate, for each replicate 5 spectra (with 256 co-averages) were acquired with a resolution of 4 cm −1 . All spectra were processed using OriginPro 2020 (OriginLab Corp., USA) software: the spectra were smoothed applying a Savitzky–Golay filter (second order, 7 pts), baselined, averaged, and normalized. Bulk ATR-FTIR analysis of the bottles and their caps is described in Supplementary Notes 3. Atomic Force Microscopy Infrared Nanospectroscopy (AFM-IR) . Analysis by a customised nanoIR3 (Bruker) with enhanced single-molecule sensitivity 32,39 was performed on IR-transparent windows of ZnSe, thoroughly cleaned with LCMS-grade water and HPLC-grade ethanol. For the analysis of standard PE, PET, and PS, 500 ppm suspensions of MPs/NPs were prepared in LCMS-grade water, Nile Red stained, and deposited on the substrate in aliquots of 0.5 µL. For real sample analysis, 0.5 µL droplets were deposited on the substrate and, once dry, rinsed by flushing 1 mL of LCMS-grade water. Prior to the AFM-IR analysis, MP/NP-rich areas on the substrates were localized by fluorescence microscopy. All measurements were performed at room temperature under controlled nitrogen atmosphere with relative humidity below 3%. Morphology maps were acquired in contact mode with a silicon gold-coated PR-EX-nIR2 (Bruker, USA) cantilever with a nominal radius of ~30 nm and an elastic constant of about 0.2 N m -1 . Line scan rate was set within 0.1-0.5 Hz, and resolution between 15 and 40 nm px -1 for the analysis of standard PS NPs and between 5-10 nm px -1 for the analysis of real samples. The AFM maps were treated and analysed in MountainSPIP software. IR spectra were collected at three locations on each particle; at each location 5 spectra were acquired and averaged. Spectra were collected with a spectral resolution of 2 cm −1 , within the range 1210-1770 cm −1 . For nano-polarimetry studies, the IR light was alternatively polarized parallel or perpendicular to the surface of incidence. Pre-processing of the spectra was performed in the built-in software Analysis Studio (version 3.17) and in OriginPro software; the spectra were smoothed applying a Savitzky–Golay filter (second order, 17 pts) and adjacent averaging (5 pt), baselined, averaged, and normalized. Declarations Data Availability All data needed to evaluate the conclusions of the paper are present in the paper and the Supplementary Information file. Other data are available from the corresponding author upon request. Source data are provided with this paper . Acknowledgments This project has received funding from the European Union's Framework Programme for Research and Innovation Horizon 2020 under the Marie Skłodowska-Curie Grant Agreement No. 860775, MONPLAS. Author contributions F.S.R., M.W.F.N., and C.V. conceived the project. C.V. and F.S.R. performed the experiments. C.V. and F. S.R. analysed the data. F.S.R, M.W.F.N., H.-G.J., and A.K.U. supervised the project and provided conceptual advices. C. V. and F.S.R. wrote the article. M.W.F.N., H.-G.J., and A.K.U. contributed to the critical revision and refinement of the manuscript. Competing interests C.V. currently works for Plastics Europe . The current association did not influence the work reported here. The research conducted in this work has been performed prior to their current employment and during their PhD studies. The remaining authors declare no competing interests. Additional information Supplementary information. 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Tip-enhanced infrared nanospectroscopy via molecular expansion force detection. Nat. Photonics 8 , 307–312 (2014). Horne, F. J., Liggat, J. J., Macdonald, W. A. & Sankey, S. W. Photo-oxidation of poly(ethylene terephthalate) films intended for photo-voltaic backsheet. J. Appl. Polym. Sci. Polym. Sci. 137 , 48623 (2020). Gardette, M. et al. Photo- and thermal-oxidation of polyethylene: Comparison of mechanisms and influence of unsaturation content. Polym. Degrad. Stab. 98 , 2383–2390 (2013). Meyns, M. et al. Multi-feature round silicon membrane filters enable fractionation and analysis of small micro- and nanoplastics with Raman spectroscopy and nano-FTIR. Anal. Methods 15 , 606–617 (2022). Li, Y., Wang, Z. & Guan, B. Separation and identification of nanoplastics in tap water. Environ. Res. 204 , (2022). Merzel, R. L. et al. Uptake and Retention of Nanoplastics in Quagga Mussels. Aquat. Nanoplastics 4 , 1800104 (2020). Liu, Y., Tan, J., Thomas, A., Ou-Yang, D. & Muzykantov, V. R. The shape of things to come: Importance of design in nanotechnology for drug delivery. Ther. Deliv. 3 , 181–194 (2012). Hoshyar, N., Gray, S., Han, H. & Bao, G. The effect of nanoparticle size on in vivo pharmacokinetics and cellular interaction. Nanomedicine 11 , 673–692 (2016). Vitali, C. et al. Quantitative image analysis of microplastics in bottled water using artificial intelligence. Talanta 266 , 124965 (2024). Vitali, C., Janssen, H. G., Ruggeri, F. S. & Nielen, M. W. F. Rapid Single Particle Atmospheric Solids Analysis Probe-Mass Spectrometry for Multimodal Analysis of Microplastics. Anal. Chem. 95 , 1395–1401 (2023). Additional Declarations Yes there is potential Competing Interest. C.V. currently works for Plastics Europe. The current association did not influence the work reported here. The research conducted in this work has been performed prior to their current employment and during their PhD studies. The remaining authors declare no competing interests. Supplementary Files Competinginterestssigned.pdf Statement of Competing Interests NPsBottledWaterSupplsubmission.docx Supplementaty Information SupplementaryVideo1.gif SI Video 1. Spatially Confined Hyper-concentration Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6719488","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":513292813,"identity":"a1dfe8fd-b154-405d-8305-66c45d36eaf9","order_by":0,"name":"Francesco Ruggeri","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYDACZhSegQ2EfkCCljQInUCCpYcJa5Fv5058XFDBkG9wfvnFxxUF5xM33D7A9gCfFoPDvJuNZ5xhsNxw402x4RmD24kbziWwG+DVwsy7TZq3jcHA4MaZNMkGg9u5G84wsEngdVgzSMs/sJb0nw0G5whrYTgM0tIA1HK+/Rhjg8EBwlrAfuE5JmEgeYOHGeiw5PqZZxjb8Dus/+zGxzw1NgZ8548//Njwx86Y7wzzMYkP+BwGARJAlGMA5TA2ENYABvzHHxCpchSMglEwCkYaAADY10yVnyYVdgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-1232-1907","institution":"Wageningen University \u0026 Research","correspondingAuthor":true,"prefix":"","firstName":"Francesco","middleName":"","lastName":"Ruggeri","suffix":""},{"id":513292814,"identity":"fca2716b-6db0-471e-9731-c213c216e341","order_by":1,"name":"Clementina Vitali","email":"","orcid":"https://orcid.org/0000-0002-4452-7213","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Clementina","middleName":"","lastName":"Vitali","suffix":""},{"id":513292815,"identity":"f8cdf940-d2d7-4145-bafb-59143c7971d8","order_by":2,"name":"Michel M.W. Nielen","email":"","orcid":"","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Michel","middleName":"M.W.","lastName":"Nielen","suffix":""},{"id":513292816,"identity":"7163b170-ab19-4e47-b7de-236b89144a15","order_by":3,"name":"Hans-Gerd Janssen","email":"","orcid":"","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Hans-Gerd","middleName":"","lastName":"Janssen","suffix":""},{"id":513292817,"identity":"b24fcbd7-0ff2-4eed-9486-4af4b5dcdeaf","order_by":4,"name":"Anna K. Undas","email":"","orcid":"https://orcid.org/0000-0003-4454-8767","institution":"Wageningen Food Safety Research","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"K.","lastName":"Undas","suffix":""}],"badges":[],"createdAt":"2025-05-21 21:20:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6719488/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6719488/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91200372,"identity":"4c321289-946d-4e69-9325-23270c273e8f","added_by":"auto","created_at":"2025-09-12 15:25:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":604828,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eUnravelling NP contamination in commercial bottled water at the single-particle level leveraging spatially confined hyper-concentration and nano-chemical imaging and spectroscopy (AFM-IR). \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e(A) NP contamination is widespread in the environment, food, and water. (B) The spatially confined hyper-concentration of water samples on hydrophobic surfaces allows high-throughput localization of NPs for AFM-IR single particle analysis. (C)\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eThe combination of AFM and IR spectroscopy, enables a multidimensional analysis of NP contamination, including toxicological relevant insights such as 3-dimensional morphology, particle size distribution, chemical identification, surface chemistry and degradation via nano-polarimetry. This figure has been designed using resources from Flaticon.com.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6719488/v1/644d323372fdbf08b029cab1.png"},{"id":91200371,"identity":"0c584605-699c-4514-aadd-219670290892","added_by":"auto","created_at":"2025-09-12 15:25:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1150111,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFluorescence-guided nano-imaging and statistical analysis of single NPs. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e(A) Schematic of the deposition of Nile Red stained PS MPs/NPs standard on a ZnSe substrate. (B) Fluorescence microscopy imaging of bare ZnSe surface before and after the deposition of PS MPs/NPs, which concentrated on the substrate after the evaporation of the liquid phase. (C) Analysis of the autofluorescence of bare ZnSe surface (mean\u0026nbsp;±\u0026nbsp;SD; n\u0026nbsp;=\u0026nbsp;3) against total fluorescence analysis of Nile Red stained PS MPs/NPs on ZnSe (mean\u0026nbsp;±\u0026nbsp;SD; n\u0026nbsp;=\u0026nbsp;3). (D) Representative AFM morphology maps of bare ZnSe before and after the deposition of PS beads. (E) Boxplot representing the AFM analysis of particle size distribution of impurities on bare ZnSe surface (n\u0026nbsp;=\u0026nbsp;9 random 10\u0026nbsp;×\u0026nbsp;10\u0026nbsp;μm areas, n\u0026nbsp;=\u003c/em\u003e\u0026nbsp;\u003cem\u003e3 independent substrates). (F) Scatter plot of PS NPs size (grey) overlayed to 3D kernel density estimation of impurity size on ZnSe bare surface (orange), with concentric orange lines representing 1\u003c/em\u003e\u003csup\u003e\u003cem\u003est\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, 2\u003c/em\u003e\u003csup\u003e\u003cem\u003end\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, 3\u003c/em\u003e\u003csup\u003e\u003cem\u003erd \u003c/em\u003e\u003c/sup\u003e\u003cem\u003equartiles, and 95% confidence interval.\u003cbr\u003e\n\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6719488/v1/f9510f1f4b84f0ca68642713.png"},{"id":91198880,"identity":"a04f8cab-a8d4-47db-80a8-fa0ea648a330","added_by":"auto","created_at":"2025-09-12 15:17:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":698364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSingle-particle chemical analysis of PS NPs standard by AFM-IR. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e(A) Schematic of the workflow for sample preparation and nano-chemical analysis. (B) Chemical structure, ATR-FTIR spectrum, and peak assignment for polystyrene (PS). Average (Avg) ± standard deviation (SD) of N = 3 independent measurements plotted in varying grayscale intensity, each with n = 5 spectra with co-averaging of 256 spectra. AFM-IR physicochemical characterization of (C) 500 nm and (D) 100 nm PS NPs, including AFM morphology map, cross section, and nano-localised IR spectra. Avg ± SD of N = 3 independent measurements plotted in varying grayscale intensity, each with n = 3 spectra. Dashed horizontal lines on maps indicate the positions where cross-sectional profiles of the analysed particles were measured. Crosses on maps indicate the location of acquisition of the AFM-IR spectra; for 500 nm nanobeads, 3 independent measurements were performed on 3 particles; while for 100 nm nanobeads, 3 independent measurements were performed on a single particle.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6719488/v1/983dfaf595617778aee47cc9.png"},{"id":91198877,"identity":"817de56c-f09a-4b7e-b9da-8d0b6805705d","added_by":"auto","created_at":"2025-09-12 15:17:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":890201,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAFM-IR single-particle chemical analysis of PE and PET irregular powders. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e(A) PE and PET standard are spiked in water and analysed by AFM-IR nano-polarimetry. ATR-FTIR spectra and peaks assignment for (B) PET and (C) PE. Average (Avg) ± standard error (SE) of N\u0026nbsp;=\u0026nbsp;3 independent measurements plotted in varying grayscale intensity, each with n\u0026nbsp;=\u0026nbsp;5 spectra with 256\u0026nbsp;co-averaging. AFM-IR physicochemical characterization of (D) PET and (E) PE standards via AFM morphology mapping and nano-localised IR spectra (Avg\u0026nbsp;±\u0026nbsp;SE, N\u0026nbsp;=\u0026nbsp;3 independent measurements, each with n\u0026nbsp;=\u0026nbsp;5 spectra at 90\u003c/em\u003e°and\u003cem\u003e n\u0026nbsp;=\u0026nbsp;5 spectra at 0\u003c/em\u003e° \u003cem\u003epolarisation). Crosses on the maps indicate the location of acquisition of the AFM-IR spectra (F) Chemical and phase imaging of PE NP showing IR absorption at 1470\u0026nbsp;cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e-1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e and absence of IR signal at 1770\u0026nbsp;cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e-1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6719488/v1/f03c28751ba133128f2fb0ea.png"},{"id":91198873,"identity":"83babd63-24c9-4007-80d6-904c60793d81","added_by":"auto","created_at":"2025-09-12 15:17:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":944533,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSpatially confined Hyper-concentration for the nano-analysis of MPs and NPs in bottled water. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e(A) Schematics of sample preparation workflow for the nano-scale analysis of MPs/NPs in bottled water. Upon deposition of a sample droplet onto the substrate, solutes and suspended particles are concentrated as the liquid phase evaporates. Due to the presence of minerals in drinking water, an additional step was introduced to rinse away the excess of salts that could hinder the analysis. The workflow was applied to process samples of (B) analytical grade pure water, (C) 50x concentrated analytical grade pure water, (D) bottled water, (E) 50x concentrated bottled water; brightfield and fluorescence images of the resulting droplet are compared to reveal MP/NP-rich areas. (F) Total fluorescence analysis of Nile Red stained droplet of analytical grade pure water, 50x concentrated analytical grade pure water, bottled water, 50x concentrated bottled water, the error bars represent sensitivity error.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6719488/v1/d71f29fd9f240c60e949b858.png"},{"id":91198874,"identity":"e18c8861-be17-4dab-a1ae-fb40cc1f0c0f","added_by":"auto","created_at":"2025-09-12 15:17:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2343534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePhysicochemical analysis of NPs isolated from bottled water. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e(A) Schematics of the workflow for the analysis of MPs/NPs in bottled water. MPs/NPs are hyper-concentrated in a spatially localised area, then localised by fluorescence microscopy and analysed by nano-imaging, -chemical, and -polarimetry analysis by AFM-IR. (B) Nile Red stained droplet of bottled water packaged in PET bottle. Fluorescence imaging reveals MP/NP-rich areas for further AFM-IR analysis. (C) In the blue box, AFM-IR spectra of fluorescent PET MP visible in the optical field of view; average (Avg) ± standard error (SE) of N\u0026nbsp;=\u0026nbsp;3 independent measurements, each with n\u0026nbsp;=\u0026nbsp;5 spectra at 90°, n\u0026nbsp;=\u0026nbsp;5 at 0° polarisation; (D) in the cyan box, AFM-IR morphology and chemical map of PET NPs, and AFM-IR nano-localised spectra thereof (Avg\u0026nbsp;±\u0026nbsp;SE, N\u0026nbsp;=\u0026nbsp;3 independent measurements, each with n\u0026nbsp;=\u0026nbsp;5 spectra at 90° and n\u0026nbsp;=\u0026nbsp;5 spectra at 0° polarisation). (E) Chemical structure of High-Density Polyethylene (HDPE) defined by linear polymer chains of ethylene with minimal branching. (F) ATR-FTIR spectra and peaks assignment of Bio-HDPE bottle and its cap (Avg\u0026nbsp;±\u0026nbsp;SE, N\u0026nbsp;=\u0026nbsp;3 independent measurements plotted in varying grayscale intensity, each with n\u0026nbsp;=\u0026nbsp;5 spectra with 256 co-averaging). (G) AFM-IR NP analysis of water packaged in Bio-HDPE bottle including morphology and chemical imaging showing IR absorption at 1474\u0026nbsp;cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e-1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e and absence of IR signal at 1738\u0026nbsp;cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e-1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, and AFM-IR nano-localised spectra thereof; morphology map and AFM-IR spectra of HDPE fibre(Avg\u0026nbsp;±\u0026nbsp;SE, N\u0026nbsp;=\u0026nbsp;3 independent measurements, each with n\u0026nbsp;=\u0026nbsp;5 spectra at 90° and n\u0026nbsp;=\u0026nbsp;5 at 0° polarisation). Crosses on the maps indicate the location of acquisition of the AFM-IR nano-localised spectra.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6719488/v1/fc93294fcf06eedea44549af.png"},{"id":91200790,"identity":"bd49de09-99d7-46be-8f75-5bf247927c11","added_by":"auto","created_at":"2025-09-12 15:33:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7274791,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6719488/v1/a38ded3f-eeb6-445a-897e-bacc5704d966.pdf"},{"id":91200373,"identity":"44846f92-3c87-44ab-af72-8ac8d8c25a05","added_by":"auto","created_at":"2025-09-12 15:25:28","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":367341,"visible":true,"origin":"","legend":"Statement of Competing Interests","description":"","filename":"Competinginterestssigned.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6719488/v1/b6a5cdaf0d915f809bb559c7.pdf"},{"id":91198879,"identity":"70cb3243-df1b-4a6c-a43c-b37320358561","added_by":"auto","created_at":"2025-09-12 15:17:28","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3727079,"visible":true,"origin":"","legend":"Supplementaty Information","description":"","filename":"NPsBottledWaterSupplsubmission.docx","url":"https://assets-eu.researchsquare.com/files/rs-6719488/v1/6e966b2dd9897e3615a919ca.docx"},{"id":91198882,"identity":"32300dd5-92bc-4835-bac0-2959efa38050","added_by":"auto","created_at":"2025-09-12 15:17:28","extension":"gif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14922160,"visible":true,"origin":"","legend":"SI Video 1. Spatially Confined Hyper-concentration","description":"","filename":"SupplementaryVideo1.gif","url":"https://assets-eu.researchsquare.com/files/rs-6719488/v1/96dc43fa95d3e9019477111f.gif"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nC.V. currently works for Plastics Europe. The current association did not influence the work reported here. The research conducted in this work has been performed prior to their current employment and during their PhD studies. The remaining authors declare no competing interests.","formattedTitle":"Single Nanoplastics Detection and Nano-Chemical Analysis of Surface Degradation in Commercial Bottled Water","fulltext":[{"header":"Introduction ","content":"\u003cp\u003eNanoplastics (NPs) – plastic particles smaller than 1\u0026nbsp;mm\u003csup\u003e1\u003c/sup\u003e– are emerging contaminants generated by the degradation of larger plastics debris\u003csup\u003e2\u003c/sup\u003e, including microplastics (MPs) measuring less than 5\u0026nbsp;mm\u003csup\u003e1\u003c/sup\u003e. As MPs have become widespread environmental contaminants\u003csup\u003e3,4\u003c/sup\u003e,\u0026nbsp;NPs are expected to be present wherever MPs are found\u003csup\u003e2\u003c/sup\u003e. However, data on NP occurrence remains scarce due to the analytical challenges associated with their detection and characterization in real samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe detection of MPs in food\u003csup\u003e5\u003c/sup\u003e has increased awareness of human exposure\u003csup\u003e6\u003c/sup\u003e. The potential concurrent presence of NPs is of particular concern because their sub-micrometer size and increased surface-to-volume ratio may facilitate their penetration across biological membranes, including the blood-brain barrier\u003csup\u003e7\u003c/sup\u003e. Studies involving rodent models have shown that NPs ingested through spiked water or feed can reach various organs, especially the brain\u003csup\u003e8,9\u003c/sup\u003e. Recently, MPs and NPs have also been detected in human brains, with increased concentration in decedents with diagnosed dementia\u003csup\u003e10\u003c/sup\u003e.\u0026nbsp;Increasing evidences of NP neurotoxicity link them to oxidative stress, inhibition of acetylcholinesterase activity, cerebral thrombosis, and neurological dysfunction\u003csup\u003e11,12\u003c/sup\u003e.\u0026nbsp;Compared to MPs,\u0026nbsp;NPs exhibit increased capacity to accumulate in brain tissues, disrupt protein folding, and initiate the aggregation of amyloid proteins, a key molecular process in the onset of neurodegenerative diseases such as Alzheimer’s and Parkinson’s\u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe analysis of NPs poses significant challenges, primarily due to their sub-diffraction limit size and higher surface reactivity, complicating both their isolation and their chemical identification\u003csup\u003e2,14\u003c/sup\u003e.\u0026nbsp;The\u0026nbsp;sub-micrometer\u0026nbsp;size of NPs, coupled with the dominance of Brownian motion over sedimentation, limits the effectiveness of enrichment methods like dead-end filtration and density separation, commonly used for MPs\u003csup\u003e15,16\u003c/sup\u003e.\u0026nbsp;The development of dedicated sample preparation procedures is complicated by the lack of environmentally representative reference materials\u003csup\u003e2,14\u003c/sup\u003e.\u0026nbsp;Engineered nanobeads, the only commercial NP analytical standards,\u0026nbsp;do not reflect the heterogeneity of true-life NPs originating from diverse fragmentation pathways\u003csup\u003e2,16\u003c/sup\u003e. As a result, approaches that are developed relying on uniform synthetic particles often prove ineffective for the analysis of NPs in real samples\u003csup\u003e16\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn principle, several analytical techniques have the sensitivity to detect NPs\u003csup\u003e15\u003c/sup\u003e. Nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) allow measuring NPs size distribution and concentration in liquid, but provide only bulk population data. NP analysis at single particle level can be achieved with electron microscopy (EM) and atomic force microscopy (AFM), which respectively yield 2-D and 3-D imaging with sub-nanometre resolution. These techniques yet lack chemical specificity, thus have been primarily applied to monitor NPs released during the controlled degradation of plastic items\u003csup\u003e17–24\u003c/sup\u003e. Pyrolysis gas chromatography mass spectrometry (Py-GC/MS) offers chemical identification of polymers based on their thermal degradation products, still providing only bulk mass-based quantitative results of the overall MP and NP contamination\u003csup\u003e25,26\u003c/sup\u003e.\u0026nbsp;Nano-resolved vibrational spectroscopy techniques, such as hyperspectral stimulated Raman scattering (SRS)\u003csup\u003e27\u003c/sup\u003e and photothermal infrared spectroscopy (OPTIR)\u003csup\u003e28,29\u003c/sup\u003e, allow detecting individual NPs in the 200-500\u0026nbsp;nm range. Yet, the imaging capabilities of these methods rely on chemical mapping, hampering quantitative morphological characterization. Real NPs exhibit considerable heterogeneity in morphology and surface properties, including weathering and chemical degradation, relevant to their toxicity and fate in the human body\u003csup\u003e30,31\u003c/sup\u003e. Yet, such complexity cannot be resolved by any of the above techniques.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAFM-based infrared nanospectroscopy (AFM-IR), combining the nanoscale spatial resolution of AFM with the chemical identification capabilities of vibrational spectroscopy, allows the quantitative assessment of 3-D morphology and chemical properties of single-molecules and polymers at the nano-scale\u003csup\u003e32–35\u003c/sup\u003e. While AFM-IR provides a highly promising solution for a comprehensive multidimensional analysis of NPs, only few preliminary studies have applied this technology to detect polymer-specific IR signatures of NPs isolated from real environmental samples\u003csup\u003e36,37\u003c/sup\u003e. Leveraging the full potential of AFM-IR for the analysis of NPs remains limited by practical constraints, arising primarily from the challenges associated with sample preparation – a fundamental step – as matrix interference and surface degradation complicate the analysis of nano-sized particles with an inherent low signal-to-noise ratio, especially when measuring on non-metallic substrates\u003csup\u003e38\u003c/sup\u003e. Due to the time-intensive nature of nanospectroscopy techniques, maximizing analyte concentration within a confined spatial area is critical for efficient measurements. Yet, this is particularly difficult for NPs, as conventional enrichment strategies are not readily transferrable to the nanoscale.\u003c/p\u003e\n\u003cp\u003eHere, we develop a framework for the single particle physicochemical characterization in commercial bottled water of NPs as small as 30 nm. To enhance detection efficiency, we leverage spatially confined hyper-concentration on hydrophobic surfaces and perform fluorescence-guided nano-imaging and -chemical analysis using our recently developed single-molecule AFM-IR\u003csup\u003e39\u003c/sup\u003e (\u003cstrong\u003eFig.\u0026nbsp;1\u003c/strong\u003e). We first demonstrate our approach on standard polystyrene (PS) nano-beads, and polyethylene (PE) and poly(ethylene terephthalate) (PET) powders, spanning micro- to nano- scale. We then report the multimodal and correlative analysis of NPs isolated from real samples of bottled water, to unravel for the first time their physicochemical properties revealing significant surface degradation, which may critically influence their behaviour and potential toxicity. These achievements pose the basis for the development of accurate NPs standards and advancing our understanding of their interaction with and within biological systems.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eStatistical identification of NPs via fluorescence-guided nano-imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore starting the nano-analytical investigation of NPs in spiked and real water samples, we developed a fluorescence-guided nano-imaging method to prove the robust statistical discrimination of NPs against residual impurities naturally present on surfaces (\u003cstrong\u003eFig.\u0026nbsp;2\u003c/strong\u003e). Nano-imaging methods, such as AFM, require indeed the deposition of the sample on adequate substrates, and their sub-diffraction limit resolution allows to reveal the presence of residual impurities on the substrate, which are comparable in size to NPs, hence potentially interfering with their analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs a model system, we considered the nano-imaging analysis of PS nano-beads deposited on hydrophobic and IR-transparent ZnSe substrates. We investigated these substrates before and after the deposition of a mix of MP and NP analytical standards, to then allow fluorescence-guided analysis from the micro- to the nano-scale (\u003cstrong\u003eFig.\u0026nbsp;2A\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eBare ZnSe substrates showed absence of fluorescent particles (\u003cstrong\u003eFig.\u0026nbsp;2B\u003c/strong\u003e) and negligible autofluorescence (\u003cstrong\u003eFig.\u0026nbsp;2C\u003c/strong\u003e) , proving to be suitable for the observation of Nile Red stained MPs. We then observed the behaviour of MPs/NPs deposited on the substrate in water suspension. Upon evaporation of the liquid phase, the PS beads concentrated in a spatially confined area with diameter \u0026lt;1\u0026nbsp;mm (\u003cstrong\u003eFig.\u0026nbsp;2B, SI Video 1\u003c/strong\u003e), decreasing the dimension of the region of interest for high-throughput nano-imaging analysis.\u0026nbsp;The deposition of PS beads on the substrates leads to a significantly increased fluorescence signal (\u003cstrong\u003eFig.\u0026nbsp;2C\u003c/strong\u003e).\u0026nbsp;We then performed AFM mapping of ZnSe substrates before and after MPs/NPs deposition. The bare ZnSe substrate showed presence of residual nanoscale impurities (\u003cstrong\u003eFig.\u0026nbsp;2D\u003c/strong\u003e). We statistically characterized their size by collecting AFM maps of randomly selected areas (10 × 10 µm, n = 9) on independent ZnSe substrates (n = 3). The residual particles had a height of 20\u0026nbsp;±\u0026nbsp;10\u0026nbsp;nm\u0026nbsp;and cross-sectional diameters (convoluted) of 50\u0026nbsp;±\u0026nbsp;30\u0026nbsp;nm (\u003cstrong\u003eFig. 2E\u003c/strong\u003e). Compared to other hydrophobic and IR-transparent surfaces, such as ZnS, ZnSe showed lower roughness (1.8\u0026nbsp;±\u0026nbsp;0.4\u0026nbsp;nm) and a narrower size distribution of residual impurities (\u003cstrong\u003eFig.\u0026nbsp;S1-2\u003c/strong\u003e, \u003cstrong\u003eSupplementary Notes\u0026nbsp;1-2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eAt the nano-scale, the impurities on the surface were easily recognizable, which facilitates their discrimination from the NPs (\u003cstrong\u003eFig.\u0026nbsp;2D,\u0026nbsp;E\u003c/strong\u003e), which represents a key advantage for improving the efficiency of time-intensive nano-scale analysis. To evaluate the statistical relevance of the discrimination of the NPs over the impurities, we plotted the height and diameter of the PS NPs (n = 88) over a 3D kernel density of the size of the impurities (n = 1452, \u003cstrong\u003eFig.\u0026nbsp;2F\u003c/strong\u003e), which outlines a 95% confidence interval neatly praising apart NPs from the impurities on the surface.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese results proved that fluorescence-guided nano-imaging, although chemically blind, could be used to detect NPs apart from natural residual impurities already present on the surface with relevant statistical power.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStandard PS NPs in Spiked Water\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNano-imaging identified the PS NPs on the surface of analysis but was not able to provide chemical recognition. We thus next deposited on a ZnSe substrate a water suspension of PS nano-beads standards with 100\u0026nbsp;nm and 500\u0026nbsp;nm size to prove single-particle chemical analysis via AFM-IR (\u003cstrong\u003eFig.\u0026nbsp;3A\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWe preliminary performed a conventional bulk Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR) analysis of the PS standard to assign the major IR peaks of the material (\u003cstrong\u003eFig.\u0026nbsp;3B\u003c/strong\u003e, \u003cstrong\u003eFig.\u0026nbsp;S3\u003c/strong\u003e, \u003cstrong\u003eTable\u0026nbsp;S1-7\u003c/strong\u003e, \u003cstrong\u003eSI Notes\u0026nbsp;3\u003c/strong\u003e). In the spectra, we observed the typical IR absorption bands of PS – 1600, 1582, 1492, and 1452\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e – \u0026nbsp;arising from the C-C stretching vibrations in conjugated six-membered rings\u003csup\u003e40\u003c/sup\u003e. We then characterized the PS standard by AFM-IR, which allowed to acquire the 3-D morphology and nano-localized infrared spectra of single nanobeads, of the two standards with size of 500\u0026nbsp;nm (\u003cstrong\u003eFig.\u0026nbsp;3C\u003c/strong\u003e) and 100\u0026nbsp;nm (\u003cstrong\u003eFig.\u0026nbsp;3D\u003c/strong\u003e). The AFM-IR analysis showed 3-D morphology of the nanobeads in excellent agreement with information provided by the producer of the standard, and the spectra showed the typical C-C stretching of PS, and with ATR-FTIR.\u003c/p\u003e\n\u003cp\u003eThus, AFM-IR proved capable of correlating the physicochemical properties of standard PS nanobeads – such as their size, shape, and chemical identity – in spiked water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNano-Chemical Identification of PE and PET Powder MPs/NPs in Spiked Water\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next applied AFM-IR for the analysis of irregular powder of PE and PET and spiked in water (\u003cstrong\u003eFig.\u0026nbsp;4\u003c/strong\u003e). These standard materials present variable shape, size, and surface properties, thus they more closely resemble MPs/NPs typically isolated from food or environmental matrices\u003csup\u003e16\u003c/sup\u003e compared to PS micro- and nano-beads. Of note, while our work is focused on NPs analysis, AFM-IR allows also high throughput characterisation of MPs.\u003c/p\u003e\n\u003cp\u003eSuspensions of PE and PET MPs/NPs were deposited on ZnSe substrates (\u003cstrong\u003eFig.\u0026nbsp;4A\u003c/strong\u003e). Before nano-chemical analysis, the bulk spectra of PE and PET MPs/NPs spiked in water were measured by ATR-FTIR (\u003cstrong\u003eFig.\u0026nbsp;4B-C\u003c/strong\u003e, \u003cstrong\u003eFig.\u0026nbsp;S3\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;Table S1-7\u003c/strong\u003e). In the spectrum of PET (\u003cstrong\u003eFig.\u0026nbsp;4B\u003c/strong\u003e), the peak at 1712\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e was assigned to the carbon-oxygen (C=O) stretching vibration in the aryl carbonyl complex; the 1613 cm\u003csup\u003e-1\u003c/sup\u003e, 1579\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e, 1504\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e, and 1406\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e peaks to carbon-carbon (C-C) stretching vibration in the benzene ring; the peak at 1341 cm\u003csup\u003e-1\u003c/sup\u003e to the twisting vibration of the methylene-oxygen bond (O-CH\u003csub\u003e2\u003c/sub\u003e); the peak at 1241\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e to the asymmetric vibration of the ester group (C-O-C). In the spectrum of PE MPs/NPs (\u003cstrong\u003eFig.\u0026nbsp;4C\u003c/strong\u003e) a major peak at 1470\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e was assigned to methylene (CH\u003csub\u003e2\u003c/sub\u003e) scissor vibration; weaker bands at 1437\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e and 1368 cm\u003csup\u003e-1\u003c/sup\u003e were assigned to the methyl (CH\u003csub\u003e3\u003c/sub\u003e) asymmetric and symmetric deformation, respectively; and the peak at 1304\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e to the twisting vibration along the methylene (CH\u003csub\u003e2\u003c/sub\u003e) chain.\u003c/p\u003e\n\u003cp\u003eWe then pursued the sing-particle nano-chemical characterisation of PET and PE MPs/NPs by AFM-IR (\u003cstrong\u003eFig.\u0026nbsp;4D-F\u003c/strong\u003e). AFM-IR allowed to correlate the 3-D morphology of the particles via nano-imaging with their chemical identity and molecular composition via nano-localised IR spectra and chemical imaging (\u003cstrong\u003eFig.\u0026nbsp;4D-F\u003c/strong\u003e). PET NPs showed irregular shapes and a high degree of similarity of the chemical spectra with those obtained by ATR-FTIR, with major peaks of absorption related to the C=O, C=C and C-O-C molecular bonds (\u003cstrong\u003eFig. 4D\u003c/strong\u003e). This single particle multimodal morphological and chemical analysis was similarly possible for PE MPs/NPs, which showed pyramidal shape in the micro- and nano-size range, with a major IR peak associated to CH\u003csub\u003e2\u003c/sub\u003e absorption (\u003cstrong\u003eFig.\u0026nbsp;4\u003c/strong\u003e\u003cstrong\u003eE\u003c/strong\u003e). AFM-IR also allowed to correlate the morphology maps with chemical IR absorption maps of the NPs with high-resolution and sensitivity (\u003cstrong\u003eFig.\u0026nbsp;4F\u003c/strong\u003e); IR maps at 1470\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e and 1770 cm\u003csup\u003e-1\u003c/sup\u003e – where PE is expected to show intense and null absorption, respectively – proved that the chemical signal arise purely from a single NP (\u003cstrong\u003eFig.\u0026nbsp;4F\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-Particle Nano-polarimetry to detect MPs/NPs surface degradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the AFM-IR spectra of the PE MP and the PE NP (\u003cstrong\u003eFig.\u0026nbsp;S4\u003c/strong\u003e and \u003cstrong\u003eFig.\u0026nbsp;4E\u003c/strong\u003e), while both particles are dominated by the methylene absorption at 1470 cm\u003csup\u003e-1\u003c/sup\u003e, two additional broad bands with maxima of intensity at 1600\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e and 1374 cm\u003csup\u003e-1\u003c/sup\u003e are visible. These bands do not typically belong to the spectrum of pure PE, thus their identification required further considerations. As declared by the supplier, the studied PE material underwent a fluoro-oxidation during the manufacturing process, to introduce acid groups on the particle surfaces. Carboxylate salts typically exhibit absorption in the regions of 1620-1560 cm⁻¹ and 1420-1340 cm⁻¹, corresponding to the asymmetric and symmetric stretching of the carboxylate group (–COOH), respectively\u003csup\u003e40\u003c/sup\u003e. These additional bands could therefore be linked to the surface modification of the MPs/NPs.\u003c/p\u003e\n\u003cp\u003eTo substantiate the hypothesis of surface modification of our PE MPs/NPs, we exploited the nano-polarimetry capabilities of AFM-IR. Due to the electromagnetic enhancement of the optical field intensity at the apex of our gold-coated AFM tip, measurements performed irradiating the sample with p-polarized (90°) IR light result in spectra where the contribution of superficial layers is more significant\u003csup\u003e41\u003c/sup\u003e; instead, spectra acquired with s-polarized IR light (0°), without enhancement at the tip, are more sensitive to the bulk chemical properties of the particles. We thus acquired single-particle spectra of PE MPs and NPs as a function of the polarization of the IR light. The spectra showed that the intensity of absorption of the band ascribed to the surface modification at 1600\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e is stronger in the spectra acquired with surface sensitive (90°) polarization, and weaker in the spectra acquired with bulk sensitive (0°) polarization for both PE particles (\u003cstrong\u003eFig.\u0026nbsp;4E, Fig.\u0026nbsp;S4\u003c/strong\u003e). The increased presence of carboxylate compared to methylene groups on the particle surfaces confirms that the unexpected absorption band at\u0026nbsp;1600\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e and 1374 cm\u003csup\u003e-1\u003c/sup\u003e in the IR spectra arises from surface modification, in agreement with what declared by the manufacturer.\u003c/p\u003e\n\u003cp\u003eOverall, we could demonstrate that not only AFM-IR is able to chemically identify standard of spherical and irregular shape in water, spanning from the MP to the NP single particle scale, but also that this technique empowers the characterization of the surface properties of the particles, allowing the study of MP and NP weathering and chemical degradation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpatially Confined Hyper-Concentration of MPs/NPs from real sample of bottled water\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe investigation of NPs contamination in real samples of commercial bottled water presents further challenges compared to the analysis of sample spiked with standard nanobeads, as discussed here and in previous studies\u003csup\u003e2,16\u003c/sup\u003e. The estimated concentration of MPs/NPs in bottled water of 2.4\u0026nbsp;±\u0026nbsp;1.3\u0026nbsp;particle/L\u003csup\u003e27\u003c/sup\u003e is far lower than the optimal level for single-particle nano-scale analysis. Thus, hyper-concentration methods are required not only to concentrate the MPs/NPs in solution, but also to confine them in a surface of analysis that is compatible with micro-to-nano spatially resolved techniques and allows time-efficient analysis.\u003c/p\u003e\n\u003cp\u003eTo achieve spatially-confined hyper-concentration, we first pre-concentrated the samples, reducing the volume while retaining MPs/NPs, and then deposited them in order to dry within a spatially-confined surface with diameter \u0026lt;1\u0026nbsp;mm (\u003cstrong\u003eFig. 5A\u003c/strong\u003e). We achieved 50x concentrated samples using a rotary evaporator. This operation resulted as well in the concentration of the minerals naturally present in bottled water, which added further complexity to the samples once deposited. To prevent the matrix from masking or interfering with the detection of plastic particles, we performed an additional rinsing step aimed at removing excess salts. An increasingly invasive handing of real water samples comes with a higher chance of procedural contamination, thus we performed the procedure on: analytical grade pure water (\u003cstrong\u003eFig\u0026nbsp;5B\u003c/strong\u003e), analytical grade pure water concentrated by 50x (\u003cstrong\u003eFig\u0026nbsp;5C\u003c/strong\u003e); bottled water (\u003cstrong\u003eFig\u0026nbsp;5D\u003c/strong\u003e), and bottled water concentrated by 50x (\u003cstrong\u003eFig\u0026nbsp;5E\u003c/strong\u003e). \u003cstrong\u003eFig.\u0026nbsp;5B-E\u003c/strong\u003e show comparative brightfield and fluorescence images, via Nile Red staining, of the resulting spatially confined and hyper-concentrated samples, revealing the distribution of MPs/NPs by increased fluorescence intensity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA quantification of total fluorescence of each sample (\u003cstrong\u003eFig.\u0026nbsp;5F\u003c/strong\u003e) showed that negligible MPs/NPs content is present in pure water, while 50x pre-concentration allowed the enhanced detection of MPs/NPs in bottled water. Thus, the presented hyper-concentration method overall allowed to spatially confine MPs/NPs on a finite area for nano-chemical analysis while maintaining procedural contamination at a negligible level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysicochemical Identification and Degradation Analysis of NPs in real samples of bottled water\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe finally proved our framework combining spatially-confined hyper-concentration of particles suspended in water samples and fluorescence-guided nano-chemical imaging and spectroscopy able to detect and study NPs in real samples of commercial bottled water (\u003cstrong\u003eFig. 6\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eFig. S6\u003c/strong\u003e). Two bottled water samples were purchased from a local store. One was packaged in conventional PET, while the other was bottled in bio-HDPE, a bio-based high-density polyethylene derived from renewable resources such as sugarcane. After concentration by a factor of 50 and Nile Red staining, the samples were deposited onto ZnSe windows (\u003cstrong\u003eFig.\u0026nbsp;6A\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe first performed our analysis on the water sample stored in a PET bottle. Fluorescence microscopy was used to identify MP/NP-rich areas within or around the deposited sample for subsequent nano-analysis (\u003cstrong\u003eFig.\u0026nbsp;6B\u003c/strong\u003e). Despite NP analysis being the focus of the method developed in this study; the method is also suitable for MP analysis. Accordingly, we pointed the probe of the AFM-IR on a selected area of interest where MPs were localised (\u003cstrong\u003eFig. S6\u003c/strong\u003e). To identify the chemical identity of these MPs, we acquired nano-localised IR spectra, which showed two major bands relative to C=O and C-O-C molecular vibrations, aligning with the spectral signature of PET (\u003cstrong\u003eFig.\u0026nbsp;6C\u003c/strong\u003e). We further performed nano-imaging to investigate the presence of NPs around the MPs, revealing numerous nanoparticles. We thus acquired nano-localised single-particle spectra to assess their chemical identify, on NPs as small as 30 nm in height (\u003cstrong\u003eFig.\u0026nbsp;6D\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;Fig. S6\u003c/strong\u003e). The spectra showed typical absorption bands of C=O, C=C, and C-O-C stretching vibrations, thus allowing to identify them as PET, in agreement with bulk FTIR (\u003cstrong\u003eFig.\u0026nbsp;S3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe spectra of PET MPs also showed additional absorption bands, which could be associated to the photo-oxidation and degradation of PET, likely resulting from sunlight exposure during transport and storage of water bottles. Exposure to UV light provides enough energy to break chemical bonds. Oxygen, then, can react with these broken bonds to form new groups such as quinones and aldehydes. Indeed, the observed peaks corresponded to IR absorption of quinones\u003csup\u003e42\u003c/sup\u003e (C=O stretching absorption, 1690-1655 cm\u003csup\u003e-1\u003c/sup\u003e)\u003csup\u003e40\u003c/sup\u003e and aldehydes\u003csup\u003e42\u003c/sup\u003e (CH rocking vibration, 1415-1350 cm\u003csup\u003e-1\u003c/sup\u003e)\u003csup\u003e40\u003c/sup\u003e. The spectra of PET NPs also showed signs of surface degradation (\u003cstrong\u003eFig.\u0026nbsp;6D\u003c/strong\u003e). Compared to the spectra acquired by irradiating the samples with s-polarized IR light, the spectra obtained with p-polarized light present a decreased absorption intensity in correspondence to the ester COC asymmetric stretching vibration peak, which has been reported as a typical sign of PET photo-oxidation\u003csup\u003e42\u003c/sup\u003e. Furthermore, the shifts in the C=O to COC ratio suggest varying degrees of ester bond cleavage, indicating chain scission as a likely degradation pathway contributing to the formation of progressively smaller plastic particles.\u003c/p\u003e\n\u003cp\u003eWe next performed our analysis on water bottled in bio-HDPE, which also allowed to identify and characterise NPs physicochemical properties (\u003cstrong\u003eFig.\u0026nbsp;6E-G\u003c/strong\u003e). The bulk IR signature of Bio-HDPE is dominated by CH\u003csub\u003e2\u003c/sub\u003e and CH\u003csub\u003e3\u003c/sub\u003e molecular vibrations (\u003cstrong\u003eFig. 6E\u003c/strong\u003e). We further performed AFM-IR analysis, and the nanoscale morphology maps of a NP and a fibre isolated from bottled water packaged in bio-HDPE are reported, along with their spectra (\u003cstrong\u003eFig. 6G\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;Fig. S6\u003c/strong\u003e). The strong CH\u003csub\u003e2\u0026nbsp;\u003c/sub\u003escissoring absorption band and the weaker but visible symmetric deformation of methyl groups allowed to chemically identify the particles as PE. In the spectra of the particles isolated from the bio-HDPE bottle, two additional peaks at 1573 and 1422\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e were observed, which may be assigned to the asymmetric and symmetric stretch of acetate ions (C=O)\u003csup\u003e40\u003c/sup\u003e. These chemical groups are typically associated with PE oxidation\u003csup\u003e43\u003c/sup\u003e, suggesting that PE NPs in water undergo significant degradation.\u003c/p\u003e\n\u003cp\u003eWe could thus, for the first time, demonstrate the single-particle multidimensional physicochemical characterisation of NPs in commercial bottled water, including the analysis of chemical modification involving either the whole particle or just its surface. These findings provide new insights into plastic degradation mechanisms at the nanoscale and may serve as a foundation for future investigations into the surface chemistry of environmental NPs, supporting the development of more environmentally relevant NP standards.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe developed a framework combining spatially confined hyper-concentration of water samples with fluorescence-guided nano-imaging and -chemical analysis, enabling the detection and characterisation of NP at the single-particle level down to 30 nm size. Our approach overcomes the challenges posed by the sub-micrometre size of NPs and their typically low concentration in real samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe enrichment of the sample by evaporation of the liquid phase, enabled the concentration of NPs across the entire size range, in contrast to conventional filtration-based methods, which fail to isolate particles smaller than the filter pore size\u003csup\u003e44\u003c/sup\u003e. The use of Nile Red as an indicator of MP/NP-rich areas guided the localization of the NPs on the surface, enhancing the analytical throughput. While previous attempt to detect NPs in dinking water\u003csup\u003e45\u003c/sup\u003e and biota\u003csup\u003e46\u003c/sup\u003e have cited the low concentration of analytes and the time-consuming nature of nano-chemical methods as obstacles to successful NP analysis, we demonstrated that with optimized sample preparation and methodology, the full potential of AFM-IR for comprehensive NP characterization can be realized.\u003c/p\u003e\n\u003cp\u003eOur AFM-IR based approach enabled, for the first time, the simultaneous acquisition of nano-resolved 3D morphology and chemical properties of NPs in the biologically relevant size range of 10\u0026nbsp;nm to 1000\u0026nbsp;nm, following their isolation from both standard materials and real bottled water samples. Detailed morphological data are particularly significant, as the size and shape of NPs play a critical role in their transport, fate, and interaction with biological systems. Insights from drug design, which can be extended to NP research, have shown that particle size and shape determines cellular uptake efficiency and circulation time, with particles \u0026lt;10\u0026nbsp;nm being rapidly eliminated by the kidneys and those \u0026gt;200\u0026nbsp;nm being cleared from the bloodstream and accumulated in the liver and spleen\u003csup\u003e47\u003c/sup\u003e. Similarly, shape affects cellular uptake and tissue penetration\u003csup\u003e48\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, surface chemistry of nanoparticles plays a key role in their interactions with biological systems\u003csup\u003e47,48\u003c/sup\u003e, affecting particle reactivity, ability to cross biological membranes, and ultimately the potential induction of adverse biological responses. The use of nano-polarimetry allowed us to investigate the surface chemistry of MPs and NPs. To the best of our knowledge, this is the first report detailing the surface modification of NPs isolated from real samples, providing a foundation for future research into degradation mechanisms at the nanoscale. Such insights will promote the development of more environmentally relevant NP standards, which will, in turn, support advancements across multiple areas of NP research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, our approach represents a powerful platform for the comprehensive physicochemical characterization of NPs. By unlocking the full capabilities of AFM-IR, it paves the way for advancing our understanding of the environmental and health impacts of NP contamination, including the elucidation of the interactions between NPs and biological systems. Future investigations could leverage AFM-IR to explore critical questions regarding the role of ingested NPs in initiating biological responses, such as protein misfolding and aggregation, which have been implicated in the onset and progression of neurodegenerative diseases.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eChemicals and standards.\u003c/strong\u003eUltra-high molecular weight, surface-modified, polyethylene (PE) powder, with nominal size of 40-48\u0026nbsp;µm, was purchased form Sigma-Aldrich\u0026nbsp;(Schnelldorf, Germany). Poly(ethylene terephthalate) (PET) powder and Poly(hydroxy butyrate)/poly(hydroxy valerate) 2% biopolymer (PHB), both with a nominal size of 300 µm, were purchased from Goodfellow (Hamburg, Germany). Polypropylene (PP) rod (Goodfellows) and poly(lactic acid) filament (3D Jake, Austria) were grinded in-house down to the microscale with a metal file. Polystyrene (PS) analytical standards with certified particle sizes (100\u0026nbsp;nm, 500\u0026nbsp;nm, 1\u0026nbsp;µm, 2\u0026nbsp;µm, 5\u0026nbsp;µm) were purchased from Sigma-Aldrich. Ultrapure water was obtained in-house from a MilliQ Water Purification System (Merk, Darmstadt, Germany).LC-MSgrade water was purchased from Rathburn (Walkerburn, United Kingdom), HPLC-grade acetone from Actu-All Chemicals (Oss, Netherlands), and HPLC-grade ethanol from (Supelco, Darmstadt, Germany). Nile Red crystals were purchased form Sigma-Aldrich. Tween20 was purchased form Sigma Aldrich.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal samples.\u003c/strong\u003e Bottled water samples were purchased at a local store, including 500 mL PET bottle containing still mineral spring water; and 500 mL Bio-HDPE bottle containing still tap water. The entire content of each bottle was concentrated by a factor of 50 with a rotary evaporator operated at 40\u0026nbsp;°C and 45 mbar for\u0026nbsp;~8\u0026nbsp;hrs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContamination prevention.\u003c/strong\u003e To prevent environmental contamination of samples and standards, 100% cotton lab coats and particle-free nitrile gloves were worn during operations. In addition to the routine cleaning procedure, each piece of glassware was washed with ultrapure water and Tween20, rinsed with analytical grade acetone, and baked overnight at 550 °C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescence microscopy.\u0026nbsp;\u003c/strong\u003eBrightfield and fluorescence images were captured using an Olympus BX51 microscope, fitted with 4x, 10x, 20x, and 40x Olympus objectives, an Olympus SC50 RGB camera, an Olympus U-RFL-T UV lamp, and a bandpass filter with excitation wavelengths of 460-490\u0026nbsp;nm and emission wavelengths greater than 515\u0026nbsp;nm. The images were collected by built-in software cellSens; fluorescence images were processed in ImageJ (version 1.54) by subtracting the background (rolling ball radius of 25\u0026nbsp;px) and applying a median filer (radius of 5\u0026nbsp;px). For fluorescence observation, MPs/NPs were suspended in MilliQ water, tagged with Nile Red\u003csup\u003e49,50\u003c/sup\u003e, and deposited on ZnSe substrates. Protocol for total fluorescence analysis is reported in Supplementary Notes\u0026nbsp;1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAtomic force microscopy.\u003c/strong\u003e AFM maps were acquired on a MultiMode 8 AFM and its built-in software Nanoscope (version 9.7, Bruker) operating in Peak Force tapping mode and equipped with ScanAsyst-air silicon tip with a nominal radius of ~2 nm and an elastic constant of about 0.4 N\u0026nbsp;m\u003csup\u003e-1\u003c/sup\u003e. Suitability for NPs analysis was assessed for ZnSe and ZnS substrates (Crystran Limited, Poole, United Kingdom) by characterization of the bare surface and AFM imaging of standard PS NPs. For the latter, 250\u0026nbsp;ppm suspensions of PS NPs were prepared in MilliQ water. Image flattening, root mean square roughness measurements, and particle analysis were performed in MountainsSPIP (version 10, Digital Surf, France) software. Protocols and results are reported in Supplementary Notes\u0026nbsp;2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAttenuated Total Reflectance-Fourier Transform Infrared spectroscopy.\u003c/strong\u003e ATR-FTIR analysis was performed using a Bruker Tensor27 spectrometer equipped with a diamond ATR element. Spectra were collected by built-in software OPUS (version 7.8, Bruker, USA). MP suspensions at a concentration of 10% (w/w) were prepared in ultra-pure water. Following a 30 s vortex agitation, a 0.5\u0026nbsp;μL aliquot was sampled and deposited on the ATR crystal. Each suspension was analysed in triplicate, for each replicate 5 spectra (with 256 co-averages) were acquired with a resolution of 4\u0026nbsp;cm\u003csup\u003e−1\u003c/sup\u003e. All spectra were processed using OriginPro 2020 (OriginLab Corp., USA) software: the spectra were smoothed applying a Savitzky–Golay filter (second order, 7 pts), baselined, averaged, and normalized. Bulk ATR-FTIR analysis of the bottles and their caps is described in Supplementary Notes 3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAtomic Force Microscopy Infrared Nanospectroscopy (AFM-IR)\u003c/strong\u003e. Analysis by a customised nanoIR3 (Bruker) with enhanced single-molecule sensitivity\u003csup\u003e32,39\u003c/sup\u003e was performed on IR-transparent windows of ZnSe, thoroughly cleaned with LCMS-grade water and HPLC-grade ethanol. For the analysis of standard PE, PET, and PS, 500 ppm suspensions of MPs/NPs were prepared in LCMS-grade water, Nile Red stained, and deposited on the substrate in aliquots of 0.5 µL. For real sample analysis, 0.5 µL droplets were deposited on the substrate and, once dry, rinsed by flushing 1 mL of LCMS-grade water. Prior to the AFM-IR analysis, MP/NP-rich areas on the substrates were localized by fluorescence microscopy. All measurements were performed at room temperature under controlled nitrogen atmosphere with relative humidity below 3%. Morphology maps were acquired in contact mode with a silicon gold-coated PR-EX-nIR2 (Bruker, USA) cantilever with a nominal radius of ~30 nm and an elastic constant of about 0.2\u0026nbsp;N\u0026nbsp;m\u003csup\u003e-1\u003c/sup\u003e. Line scan rate was set within 0.1-0.5\u0026nbsp;Hz, and resolution between 15 and 40 nm px\u003csup\u003e-1\u003c/sup\u003e for the analysis of standard PS NPs and between 5-10 nm px\u003csup\u003e-1\u003c/sup\u003e for the analysis of real samples. The AFM maps were treated and analysed in MountainSPIP software. IR spectra were collected at three locations on each particle; at each location 5 spectra were acquired and averaged. Spectra were collected with a spectral resolution of 2 cm\u003csup\u003e−1\u003c/sup\u003e, within the range 1210-1770\u0026nbsp;cm\u003csup\u003e−1\u003c/sup\u003e. For nano-polarimetry studies, the IR light was alternatively polarized parallel or perpendicular to the surface of incidence. Pre-processing of the spectra was performed in the built-in software Analysis Studio (version\u0026nbsp;3.17) and in OriginPro software; the spectra were smoothed applying a Savitzky–Golay filter (second order, 17\u0026nbsp;pts) and adjacent averaging (5\u0026nbsp;pt), baselined, averaged, and normalized.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data needed to evaluate the conclusions of the paper are present in the paper and the Supplementary Information file. Other data are available from the corresponding author upon request. Source data are provided with this paper\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project has received funding from the European Union\u0026apos;s Framework Programme for Research and Innovation Horizon 2020 under the Marie Skłodowska-Curie Grant Agreement No. 860775, MONPLAS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eF.S.R., M.W.F.N., and C.V. conceived the project. C.V. and F.S.R. performed the experiments. C.V. and F. S.R. analysed the data. F.S.R, M.W.F.N., H.-G.J., and A.K.U. supervised the project and provided conceptual advices. C. V. and F.S.R. wrote the article. M.W.F.N., H.-G.J., and A.K.U. contributed to the critical revision and refinement of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.V. currently works for \u003cem\u003ePlastics Europe\u003c/em\u003e. The current association did not influence the work reported here. The research conducted in this work has been performed prior to their current employment and during their PhD studies. The remaining authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information.\u003c/strong\u003e Supplementary materials are available within the separate file.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cem\u003eInternational Organization for Standardization. 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Chem.\u003c/em\u003e \u003cstrong\u003e95\u003c/strong\u003e, 1395\u0026ndash;1401 (2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6719488/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6719488/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Nanoplastics (NPs) are emerging as widespread environmental and food contaminants, posing significant concerns because of their potential to penetrate biological membranes, accumulate in tissues, and induce toxic effects. For understanding the impact of NPs on human health, a detailed characterization of their complex physicochemical properties is required. Yet, current analytical methods either lack single-particle spatial resolution, sensitivity, or specificity to detect and analyse NPs contaminating real samples. Here, for the first time, we detect and perform a nano-analytical characterisation of single NPs isolated from commercial drinking water as small as 30 nm; combining spatially confined hyper-concentration, fluorescence-guided nano-imaging, and chemical analysis via infrared nanospectroscopy. Our novel approach offers high-throughput single-particle analysis in real samples, enabling a multimodal characterisation of their 3D morphology, size, chemical identity, and surface degradation. This work paves the way to detect and analyse NPs in complex food matrices and biological systems, to study their interactions, fate, and toxicity.","manuscriptTitle":"Single Nanoplastics Detection and Nano-Chemical Analysis of Surface Degradation in Commercial Bottled Water","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 15:17:24","doi":"10.21203/rs.3.rs-6719488/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-food","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"natfood","sideBox":"Learn more about [Nature Food](http://www.nature.com/natfood/)","snPcode":"","submissionUrl":"","title":"Nature Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c693296a-4c04-413e-912f-37d6c9622ae7","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":54531024,"name":"Physical sciences/Chemistry/Analytical chemistry/Infrared spectroscopy"},{"id":54531025,"name":"Earth and environmental sciences/Environmental sciences/Environmental impact"},{"id":54531026,"name":"Earth and environmental sciences/Environmental sciences/Environmental chemistry"},{"id":54531027,"name":"Physical sciences/Nanoscience and technology/Techniques and instrumentation/Imaging techniques"}],"tags":[],"updatedAt":"2026-04-30T17:45:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-12 15:17:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6719488","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6719488","identity":"rs-6719488","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}