Asymmetrical Flow Field-flow Fractionation Research Articles

Determination of the Nanoscale Silica Mass Fraction by AF4/ICP-MS with Isotope Dilution Analysis Using 29Si-Enriched Silica Nanoparticles.

A methodology based on the use of asymmetrical flow field-flow fractionation (AF4) coupled to ICP-MS with size fraction-targeted isotope dilution analysis (IDA) has been developed, validated, and applied for the first time to determine the mass fraction of nanoscale silica (SiO2). For this purpose, 29Si-enriched SiO2 nanoparticles, to be used as an IDA spike/internal standard, were synthesized and characterized in-house. Double IDA was used to quantify an aqueous suspension of Stöber silica particles of similar characteristics to those of the 29SiO2 nanoparticle (NP) spike using a representative test material of natural Si isotopic composition as the calibrant. For fumed SiO2 NP in a highly complex food matrix, a methodology based on single IDA with AF4/ICP-MS using the same 29SiO2 NP spike was developed and validated. Relative expanded measurement uncertainties (k = 2) of 4% (double IDA) and 8% (single IDA) were achieved for nanoscale silica mass fractions of 5143 and 107 mg kg-1 in water suspension and food matrix, respectively. To assess the accuracy of AF4/ICP-IDMS for the characterization of SiO2 NP in a food matrix, standard addition measurements on samples spiked with Aerosil AF200, also in-house characterized for Si mass fraction, were undertaken, with an average recovery of 95.6 ± 4.1% (RSD, n = 3) obtained. The particle-specific IDA data obtained for both SiO2 NP-containing samples were also compared with that of post-AF4 channel external calibration using inorganic Si standards. The mass fractions obtained by IDA agreed well with those obtained by external calibration within their associated measurement uncertainties.

Resolving Uncertainties in the Quantification of Trace Elements within Organic-Rich Boreal Rivers for AF4-UV-ICP-MS Analysis.

Over the past few decades, asymmetric flow field-flow fractionation (AF4) has emerged as a robust technique for the separation of colloid-associated trace elements (TEs) in aqueous samples. Nevertheless, little is known about potential artifacts and how to control them when measuring the concentrations of colloid-associated elements at low (μg L-1) or ultralow concentrations (ng L-1) using AF4-UV-ICP-MS. Water from a boreal river was selected as a challenging test material due to its high concentrations of dissolved organic matter (DOM) and Fe-rich colloids. These colloids are expected to be significant contributors to artifact occurrence, even in a metal-free, ultraclean laboratory. The results show that the adsorption of Mn, Co, Ni, Cu, and Pb onto acid-cleaned, non-channel surfaces (such as connection tubing and autosampler) accounted for up to 48% of TE loss. These losses on non-channel surfaces also represent potential sources of cross-contamination for Co, Ni, Cu, and Pb. New, uncleaned poly(ether sulfone) membranes are also sources of contamination for Ni and Cu. Analytical bias may exist in the measured concentrations of TEs, primarily due to the potential carryover of weakly adsorbed TEs (e.g., Ni and Cu) on the system surfaces by colloids in the samples (e.g., DOM). On the other hand, colloids in the samples can also act to gradually remove contaminants from the surfaces. For these types of DOM-rich waters, preconditioning the AF4 system using 40 mg C L-1 of Suwannee River Natural Organic Matter (SRNOM, pH = 7) is recommended to mitigate the impact of membrane fouling and carryover. A comprehensive strategy for minimizing instrumental artifacts is presented and discussed.

Comprehensive analysis of colloid formation, distribution, and properties of monovarietal red wines using asymmetrical flow field-flow fractionation with online multidetection

Red wine colloids, crucial in determining wine quality and stability, are understudied due to inadequate techniques for studying them effectively in the natural wine environment. Recently, Asymmetrical Flow Field-flow Fractionation (AF4) with online multidetection has emerged as a novel analytical tool for quantifying, fractionating, and characterizing red wine colloids in their native state. This study aimed to characterize the colloidal composition of 24 monovarietal Italian wines produced without filtration, oak contact, fining treatments, malolactic fermentation, macerating enzymes or ageing on yeast lees. AF4 analysis allowed quantification and characterization of wine colloids based on light scattering signal (MALS; gyration radius − Rg), size (hydrodynamic radius – Rh) and absorbance (A280 & A520 nm).The results showed that each wine contained up to five distinct colloids’ populations, varying in size and gyration radii. Despite possessing very similar Rh, most colloids exhibited great differences in compactness, as indicated by their varying Rg values. Comparing the A280 signal of whole wines to those of wines containing only species larger than 5 kDa (considered colloids) allowed to calculate the percentage of molecules involved in colloidal particles assembly, ranging from 1 to 44 % of the total A280 absorbing compounds, reflecting the diversity among wines. The A520 signal indicated the presence of polymeric pigments in the colloidal fraction. Notably, colored colloids all had Rg > 20 nm, indicating their association with other colloidal-forming compounds. This observation led to the conclusion that, apart from free anthocyanins and polymeric pigments, the color of red wines is also due to colloidal particles formed by the latter bound to proteins, with their quantity being highly variable across wines of different origin. These findings, which highlight the fundamental role of proteins in shaping the colloidal status of red wines, were utilized to propose an updated hypothetical model for colloidal aggregation in red wine.

Organic-mineral colloids regulate the migration and fractionation of rare earth elements in groundwater systems impacted by ion-adsorption deposits mining in South China

Ion-adsorption rare earth element (REE) deposits distributed in the subtropics provide a rich global source of REEs, but in situ injection of REEs extractant into the mine can result in leachate being leaked into the surrounding groundwater systems. Due to the lack of understanding of REE speciation distribution, particularly colloidal characteristics in a mining area, the risks of REEs migration caused by in situ leaching of ion-adsorption REE deposits has not been concerned. Here, ultrafiltration and asymmetric flow field-flow fractionation coupled with inductively coupled plasma mass spectrometry (AF4-ICP-MS) were integrated to characterize the size and composition of REEs in leachate and groundwater from mining catchments in South China. Results show that REEs were associated with four fractions: 1) the <1 kDa fraction including dissolved REEs; 2) the 1 – 100 kDa nano-colloidal fraction containing organic compounds; 3) the 100 kDa – 220 nm fine colloids including organic-mineral (Fe, Mn and Al (oxy)hydroxides and clay minerals); 4) the >220 nm coarse colloids and acid soluble particles (ASPs) comprising minerals. Influenced by the ion exchange effect of in situ leaching, REEs in leachate were mostly dissolved (79 %). The pH of the groundwater far from the mine site was increased (5.8 – 7.3), the fine organic-mineral colloids (46 % – 80 %) were the main vectors of transport for REEs. Further analysis by AF4 revealed that the fine colloids can be divided into mineral-rich (F1, 100 kDa – 120 nm) and organic matter-rich (F2, 120 – 220 nm) populations. The main colloids associated with REEs shifted from F1 (64 % ∼ 76 %) to F2 (50 % ∼ 52 %) away from the mining area. For F1 and F2, the metal/C molar ratio decreased away from the mining area and middle to heavy REE enrichment was presented. According to the REE fractionation, organic matter was the predominant component capable of binding REEs in fine colloids. Overall, our results indicate that REEs in the groundwater system shifted from the dissolved to the colloidal phase in a catchment affected by in situ leaching, and organic-mineral colloids play an important role in facilitating the migration of REEs.

Impact of Protein Nanoparticle Shape on the Immunogenicity of Antimicrobial Glycoconjugate Vaccines.

Protein self-assembling nanoparticles (NPs) can be used as carriers for antigen delivery to increase vaccine immunogenicity. NPs mimic the majority of invading pathogens, inducing a robust adaptive immune response and long-lasting protective immunity. In this context, we investigated the potential of NPs of different sizes and shapes-ring-, rod-like, and spherical particles-as carriers for bacterial oligosaccharides by evaluating in murine models the role of these parameters on the immune response. Oligosaccharides from Neisseria meningitidis type W capsular polysaccharide were conjugated to ring-shape or nanotubes of engineered Pseudomonas aeruginosa Hemolysin-corregulated protein 1 (Hcp1cc) and to spherical Helicobacter pylori ferritin. Glycoconjugated NPs were characterized using advanced technologies such as High-Performance Liquid Chromatography (HPLC), Asymmetric Flow-Field Flow fractionation (AF4), and Transmission electron microscopy (TEM) to verify their correct assembly, dimensions, and glycosylation degrees. Our results showed that spherical ferritin was able to induce the highest immune response in mice against the saccharide antigen compared to the other glycoconjugate NPs, with increased bactericidal activity compared to benchmark MenW-CRM197. We conclude that shape is a key attribute over size to be considered for glycoconjugate vaccine development.

Changes in cargoes of platelet derived extracellular vesicles heterogeneous subpopulations induced by PM0.1——Undisclosed cardiovascular injury communication mechanism

Epidemiological evidence has indicated a closely link between PM0.1 exposure and the incidence rate of cardiovascular diseases. This study explores the underlying communication roles of platelet-derived extracellular vesicles (PEVs) heterogeneous subpopulations in cardiovascular injury. PEVs and PMEVs which were extracted from platelet-rich plasma (PRP) un-exposure or exposure to PM0.1 by TIM4 affinity beads. By optimizing separation conditions, replacing pipelines, and resetting injection procedures, Asymmetric flow field-flow fractionation (AF4) was employed to separate, purify, characterize, and enrich PEVs and PMEVs heterogeneous subpopulations (small PEVs, PEVs-S/PMEVs-S: <100 nm; medium PEVs, PEVs-M/PMEVs-M: 100–200 nm; and large PEVs, PEVs-L/PMEVs-L: >200 nm). The results showed that the cargoes of PMEVs heterogeneous subpopulations which were released by PRP stimulated by PM0.1 were changed obviously. Moreover, compared with PEVs, PMEVs can lead to a decrease in the survival rate of Human Umbilical Vein Endothelial Cells (HUVECs). In PMEVs-S subpopulations, the alterations of lipids associated with membrane fusion and cell signaling transport (such as PC, Cer), as well as miRNAs related to inflammation, angiogenesis, and migration (miR-223, miR-22, miR-126, and miR-150), are similar to those in PMEVs-M subpopulations but distinct from PMEVs-L subpopulations. This study revealed the diverse communication mechanisms underlying PM0.1-induced cardiovascular injury, thereby offering potential avenues for the development of new biomarkers and therapeutic targets.

Nanobody-mediated targeting of zinc phthalocyanine with polymer micelles as nanocarriers

Photodynamic therapy (PDT) is a suitable alternative to currently employed cancer treatments. However, the hydrophobicity of most photosensitizers (e.g., zinc phthalocyanine (ZnPC)) leads to their aggregation in blood. Moreover, non-specific accumulation in skin and low clearance rate of ZnPC leads to long-lasting skin photosensitization, forcing patients with a short life expectancy to remain indoors. Consequently, the clinical implementation of these photosensitizers is limited. Here, benzyl-poly(ε-caprolactone)-b-poly(ethylene glycol) micelles encapsulating ZnPC (ZnPC-M) were investigated to increase the solubility of ZnPC and its specificity towards cancers cells. Asymmetric flow field-flow fractionation was used to characterize micelles with different ZnPC-to-polymer ratios and their stability in human plasma. The ZnPC-M with the lowest payload (0.2 and 0.4% ZnPC w/w) were the most stable in plasma, exhibiting minimal ZnPC transfer to lipoproteins, and induced the highest phototoxicity in three cancer cell lines. Nanobodies (Nbs) with binding specificity towards hepatocyte growth factor receptor (MET) or epidermal growth factor receptor (EGFR) were conjugated to ZnPC-M to facilitate cell targeting and internalization. MET- and EGFR-targeting micelles enhanced the association and the phototoxicity in cells expressing the target receptor. Altogether, these results indicate that ZnPC-M decorated with Nbs targeting overexpressed proteins on cancer cells may provide a better alternative to currently approved formulations.

Pilot plant approach combining photocatalysis and adsorption for antibiotics removal from slaughterhouse and urban wastewater treatment plant effluents

The main objective of this research is to perform a real case-study of antibiotic decontamination from real slaughterhouse and wastewater effluents by applying a sequential treatment of TiO2/UV–vis photocatalysis and adsorption. More precisely, the removal of five classes of antibiotics: enrofloxacin, sulfadiazine, trimethoprim, azithromycin, amoxicillin and amoxicillin degradation products was studied in an 80 L/h photocatalytic and adsorption pilot-scale plant over multiple cycles, operating in semi-continuous mode. The results exhibited significant removal rates, ranging from 77% to 100% for the slaughterhouse effluent, and 61–89% for the wastewater treatment plant effluent, thus demonstrating that the treatment is more effective when it is applied directly in the emission source, previous to antibiotic dilution in the municipal collector. Using the two processes sequentially results in greater efficiency than when they are used in isolation. After photocatalysis, antibiotic degradation products are easily adsorbed, since they have more affinity for the adsorbent, and the presence of competing compounds decreases considerably. After applying several cycles of treatment, the adsorption performance remains almost constant. By contrast, the photocatalysis performance decreased, which was attributed to catalyst agglomeration determined by Asymmetric Flow Field-Flow Fractionation (AF4) coupled with Dynamic Light Scattering (DLS) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS).

Soil colloids as binding agents in the formation of soil microaggregates in wet-dry cycles: A case study for arable Luvisols under different management

In the hierarchical model of soil aggregates, small soil microaggregates (small SMA; <20 μm) are often considered to be fundamental building units at the micron scale. Below which, soil colloids (<1 µm) have recently been proposed as binding agents of (micro)aggregates. However, the way in which soil colloids contribute to the formation and stability of soil micro- and macroaggregates remains largely unknown. For clarification, we evaluated potential impacts of the colloidal content, particularly the <450 nm colloids, on the aggregation of small SMA. Free water stable small SMA and <450 nm colloids were isolated from Ap-horizons of Stagnic Luvisols under different management (cropped and bare fallow). The size-resolved elemental composition of the <450 nm colloids was analyzed by asymmetric flow field-flow fractionation in combination with an inductively coupled plasma mass spectrometer and an organic carbon detector. To vary the colloidal content in small SMA, (1) suspensions containing different amounts of <450 nm colloids were added in small SMA, or (2) <1 µm colloids were removed from small SMA by centrifugation. In the maximum colloidal addition treatment, the mass ratios of added colloids to small SMA were 3.0 and 5.1 wt% for the cropped and bare fallow soil samples, respectively. Aggregation of small SMA with different colloidal amounts was performed in three successive wet-dry cycles. Afterwards, the size distribution of the resulting aggregates was measured by laser diffraction. Our results indicated that, in wet-dry cycles, colloids were important binding agents for the formation of SMA. Their presence, especially those <450 nm, was likely to support the formation of solid bridges during drying at particle contacts of 1–10 µm small SMA, favoring hereby SMA build-up in a relatively small size range of 1–40 µm. In contrast, the absence of <1 μm colloids in small SMA led to a preferential generation of relatively large aggregates in wet-dry cycles, i.e., typically with sizes >40 μm up to 1700 μm in maximum. Our study on aggregation in wet-dry cycles revealed that the colloidal content has a controlling effect on the size distribution of resulting aggregates by acting as a binding agent and provides hereby new insights into the evolvement of aggregate hierarchy in soils.

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device.

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

Characterization of molar mass and conformation of relevant (non-)starch polysaccharides in cereal-based beverages

Arabinoxylans, β-glucans, and dextrins influence the brewing industry's filtration process and product quality. Despite their relevance, only a maximum concentration of β-glucans is recommended. Nevertheless, filtration problems are still present, indicating that although the chemical concentration is essential, other parameters should be investigated. Molar mass and conformation are important polymer physical characteristics often neglected in this industry. Therefore, this research proposes an approach to physically characterize enzymatically isolated beer polysaccharides by asymmetrical flow field-flow fractionation coupled to multi-angle light scattering and differential refractive index detector. Based on the obtained molar masses, root-mean-square radius (rrms from MALS), and hydrodynamic radius (rhyd), conformational properties such as apparent density (ρapp) and rrms/rhyd can be calculated based on their molar mass and size. Consequently, the ρapp and rrms/rhyd behavior hints at the different structures within each polysaccharide. The rrms/rhyd 1.2 and high ρapp values on low molar mass dextrins (1–2·105 g/mol) indicate branches, while aggregated structures at high molar masses on arabinoxylans and β-glucans (2·105 –6·106 g/mol) are due to an increase of ρapp and a rrms/rhyd (0.6–1). This methodology provides a new perspective to analyze starch and non-starch polysaccharides in cereal-based beverages since different physical characteristics could influence beer's filtration and sensory characteristics.

Are Aptamers Really Promising as Receptors for Analytical Purposes? Insights into Anti-Lysozyme DNA Aptamers through a Multitechnique Study.

Aptamers are recognition elements increasingly used for the development of biosensing strategies, especially in the detection of proteins or small molecule targets. Lysozyme, which is recognized as an important biomarker for various diseases and a major allergenic protein found in egg whites, is one of the main analytical targets of aptamer-based biosensors. However, since aptamer-based strategies can be prone to artifacts and data misinterpretation, rigorous strategies for multifaceted characterization of the aptamer-target interaction are needed. In this work, a multitechnique approach has been devised to get further insights into the binding performance of the anti-lysozyme DNA aptamers commonly used in the literature. To study molecular interactions between lysozyme and different anti-lysozyme DNA aptamers, measurements based on a magneto-electrochemical apta-assay, circular dichroism spectroscopy, fluorescence spectroscopy, and asymmetrical flow field-flow fractionation were performed. The reliability and versatility of the approach were proved by investigating a SELEX-selected RNA aptamer reported in the literature, that acts as a positive control. The results confirmed that an interaction in the low micromolar range is present in the investigated binding buffers, and the binding is not associated with a conformational change of either the protein or the DNA aptamer. The similar behavior of the anti-lysozyme DNA aptamers compared to that of randomized sequences and polythymine, used as negative controls, showed nonsequence-specific interactions. This study demonstrates that severe testing of aptamers resulting from SELEX selection is the unique way to push these biorecognition elements toward reliable and reproducible results in the analytical field.

Copper Speciation in Wine Growing-Drain Waters: Mobilization, Transport, and Environmental Diffusion

Copper (Cu) has been used to treat vines for a long time, which has led to its accumulation in vineyard soils. In the present work, the mobilization of copper from these soils and its transport, and diffusion outside the plots by drain water were investigated. For this, the distribution of copper between the dissolved and colloidal phases, and within the colloidal phase, of these waters was determined using an investigation strategy based on the coupling between a size separation technique, asymmetric flow field-flow fractionation, and several detectors. First, the total copper concentrations in water from different drains were monitored over a period of 2 years: Cu was mainly found in the fraction of &lt; 450 nm. Then, the distribution of copper on the size continuum was more closely studied in water from one of the drains, sampled over a winter period. Between 45 and 75% of Cu was found in the 2–450 nm colloidal fraction. The &lt;450 nm colloidal phase of the drain waters was found to be mainly composed of humic acids (~15 to 60 mg L−1) and clay-rich particles (~100 to 650 mg (Al) L−1). These particles also contained (hydr)oxides of iron and manganese. The concentrations of Fe and Mn were approximately 100 to 200 times lower than those of Al. The majority of humic acids had an apparent molar mass of ≤ 10 kDa. They were distributed along the size continuum: (i) in a population with an average size of ~20 nm, probably consisting of supramolecular entities, and (ii) associated with clay-rich particles with a size of ~120–200 nm. Copper was found to be complexed with humic acids and associated with clays via clay-humic complexes. Copper mobilization from the soil to the water and its transport to the drain water appeared governed by the soil humidity level and the rainfall.

Determination and Characterization of Gold Nanoparticles in Liquor Using Asymmetric Flow Field-Flow Fractionation Hyphenated with Inductively Coupled Plasma Mass Spectrometry.

The EU has approved the usage of gold as a food additive (E175) and it has been applied in numerous foods for coloring and decoration purposes. Different from the general assumption that edible gold is mainly present in the form of flakes or external coating in foods, this work demonstrated that gold nanoparticles (Au NPs) can be released from gold flakes and extracted under optimized conditions. To support future risk assessment associated with the exposure of Au NPs to human health, an effective approach was established in this study for both size characterization and mass determination of Au NPs released in a commercial gold-containing liquor using Asymmetric Flow Field-flow Fractionation (AF4) hyphenated with Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Our results showed that no Au NPs were detected in the original liquor product and only after ultrasonication for several minutes did Au NPs occur in the ultrasound-treated liquor. Particularly, Au NPs released in the liquor can be well extracted after 100-fold enrichment of gold flakes and the subsequent ultrasonication for 25 min. Size characterization of Au NPs was conducted by AF4-ICP-MS under calibration with Au NP standards. The gold particle sizes detected ranged from 8.3-398.0 nm and the dominant size of the released Au NPs was around 123.7 nm in the processed liquor. The mass concentration of gold particles determined in the liquor sample with gold flakes concentrated and subsequently sonicated was 48.1 μg L-1 by pre-channel calibration and the overall detection recoveries ranged over 82-95%. For the comparison control samples without ultrasonication, there was no detection of Au NPs. The established method was demonstrated to be useful for monitoring Au NPs in liquor and is possibly applied to other similar foodstuffs.

Nanoparticle Size Distribution and Stability Assessment Using Asymmetric-Flow Field-Flow Fractionation.

Nanomaterials are inherently polydisperse. Traditional techniques, such as the widely used batch-mode dynamic light-scattering (DLS) analysis, are not ideal nor thoroughly descriptive enough to define the full complexity of these materials. Asymmetric-flow field-flow fractionation (AF4) with various in-line detectors, such as ultraviolet-visible (UV-vis), multi-angle light scattering (MALS), refractive index (RI), and DLS, is an alternative technique that can provide flow-mode analysis of not only size distribution but also shape, drug release/stability, and protein binding.

An in-depth physicochemical investigation of drug-loaded core-shell UiO66 nanoMOFs.

Nanosized UiO66 are among the most studied MOF materials. They have been extensively applied in various areas, such as catalysis, gas absorption, electrochemistry, chemical sensing, and biomedical applications. However, the preparation of stable nano-sized UiO66 for drug delivery applications is challenging because of the high tendency of UiO66 to aggregate during storage. To address this issue, we coated UiO66 with oligomers made of crosslinked cyclodextrins. The coated UiO66 exhibited a good stability upon storage for more than three weeks, even for low quantities of coating materials. The resulting core-shell UiO66 were characterized using a set of complementary methods including microscopies, spectroscopies, X-ray diffraction, and thermogravimetric investigations. Size distribution was assessed by orthogonal methods. Cisplatin was loaded in the core-shell nanoparticles, followed by an in-depth analysis by asymmetric flow field-flow fractionation (AF4) hyphenated with inductively coupled plasma-mass spectrometry (ICP-MS). This method combines the extremely high elemental selectivity and ultratrace detection limits of mass spectrometry with the capacity of AF4 to differentiate the diverse populations present in the sample. Free cisplatin and UiO66-associated cisplatin could be well separated by AF4. AF4-ICP-MS/MS analysis provided the exact drug loading, without the need of separating the nanoparticles from their suspension media. These data suggest the potential of AF4-ICP-MS/MS in the optimization of drug delivery systems.

Assessment of Protein Binding Using Asymmetric-Flow Field-Flow Fractionation Combined with Multi-angle Light Scattering and Dynamic Light Scattering.

Asymmetric-flow field-flow fractionation (AF4) is a valuable tool to separate and assess different size populations in nanotherapeutics. When coupled with both static light scattering and dynamic light scattering, it can be used to qualitatively assess protein binding to nanoparticles by comparing the shape factors for both non-plasma-incubated samples and plasma-incubated samples. The shape factor is defined as the ratio of the derived root mean square radius (bystatic light scattering) to the measured hydrodynamic radius (bydynamic light scattering). The shape factor gives an idea of where the center of mass lies in a nanoparticle, and any shift in the shape factor to larger values is indicative of a mass addition to the periphery of the nanoparticle and suggests the presence of protein binding. This protocol will discuss how to set up an experiment to assess protein binding in nanoparticles using AF4, multi-angle light scattering (MALS), and dynamic light scattering (DLS).

Influence of artificial digestion on characteristics and intestinal cellular effects of micro-, submicro- and nanoplastics

The production of plastics is rising since they have been invented. Micro, submicro- and nanoplastics are produced intentionally or generated by environmental processes, and constitute ubiquitous contaminants which are ingested orally by consumers. Reported health concerns include intestinal translocation, inflammatory response, oxidative stress and cytotoxicity. Every digestive milieu in the gastrointestinal tract does have an influence on the properties of particles and can cause changes in their effect on biological systems. In this study, we subjected plastic particles of different materials (polylactic acid, polymethylmethacrylate, melamine formaldehyde) and sizes (micro- to nano-range) to a complex artificial digestion model consisting of three intestinal fluid simulants (saliva, gastric and intestinal juice). We monitored the impact of the digestion process on the particles by performing Dynamic Light Scattering, Scanning Electron Microscopy and Asymmetric Flow Field-Flow Fractionation. An in vitro model of the intestinal epithelial barrier was used to monitor cellular effects and translocation behavior of (un)digested particles. In conclusion, artificial digestion decreased cellular interaction and slightly increased transport of all particles across the intestinal barrier. The interaction with organic matter resulted in clear differences in the agglomeration behavior. Moreover, we provide evidence for polymer-, size- and surface-dependent cellular effects of the test particles.

Molecular characterization of a galactomannan extracted from Tara (Caesalpinia spinosa) seeds

Tara gum (TG) is a polysaccharide extracted from the seeds of a South American tree called Tara (Caesalpinia spinosa). TG is a galactomannan with many applications in the food industry, mainly as an emulsifier and stabilizer agent. In addition, it is also used in the paper and cosmetic industries. In the present study, we performed a molecular characterization based on chemical composition and physicochemical properties to understand the properties behind TG applications. TG was extracted and purified from Tara seeds distributed in different ecoregions of Bolivia. The monosaccharide composition analysis was determined by high-performance anion-exchange chromatography/pulsed amperometric detection (HPAEC-PAD). At the same time, their molecular characteristics, such as molar mass, root-mean-square radius, hydrodynamic radius, conformation, and densities, were studied by asymmetrical flow field-flow fractionation coupled to multi-angle light scattering refractive index (AF4-MALS-dRI), also the specific refractive index increment (dn/dc) was determined for the first time using AF4 for TG. The results revealed that the gum samples are galactomannans composed of mannose (Man) and galactose (Gal) in a ratio of 3.37 (Man/Gal), with an average molar mass range from 2.460 × 107 to 3.699 × 107 Da, distributed in a single population. The root-mean-square radius range from 260.4 to 281.6 nm, and dn/dc is 0.1454. The Kratky plots based on 14 scattering angles indicated that the conformation of all samples corresponds to random coil monodisperse, while their gyration radius/hydrodynamic radius ratio (ρ) is high. All these results suggest that the chains have a low branched density, consistent with the Gal/Man composition. To the best of our knowledge, we report for the first time an integrated physicochemical study of TG relevant to developing emulsifier and stabilizer formulations.

Design and advanced characterization of quercetin-loaded nano-liposomes prepared by high-pressure homogenization.

Quercetin-loaded nano-liposomes were prepared by high-pressure homogenization (HPH) at different pressures (up to 150MPa) and number of passes (up to 3) to define the best processing conditions allowing the lowest particle size and the highest encapsulation efficiency (EE). The process at 150MPa for 1 pass was the best, producing quercetin-loaded liposomes with the lowest particle size and 42% EE. Advanced techniques (multi-detector asymmetrical-flow field flow fractionation and analytical ultracentrifugation combined with transmission electron microscopy) were further used for the characterization of the liposomes which were oblong in shape (ca. 30nm). Results highlight the need for several techniques to study nano-sized, polydisperse samples. The potential of quercetin-loaded liposomes against colon cancer cells was demonstrated. Results prove that HPH is an efficient and sustainable method for liposome preparation and highlight the remarkable role of process optimisation as well as the powerfulness of advanced methodologies for the characterisation of nano-structures.