Asymmetrical Flow Field-flow Fractionation Research Articles

Size-Resolved Fluorescence Underscores Negligible Interaction of Dissolved Organic Matter During Conservative Mixing in a Large Boreal River

Although river mixing occurs widely in nature, the corresponding evolution of dissolved organic matter (DOM) composition remains poorly understood. Here, surface water samples were collected at multiple transects in the lower Athabasca River (LAR) under base-flow conditions. Asymmetric flow field-flow fractionation (AF4) coupled to online excitation-emission measurements (EEMs) and parallel factor analysis (PARAFAC) were utilized to investigate the size distribution of fluorescent DOM components during river mixing and the corresponding variation in size-resolved fluorescence. The majority of fluorescent components occurred at 0.810 and 1.170 kDa, reflecting the small size of the DOM molecules with maximum fluorescence. The loadings of fluorescence normalized to absorbance at 254 nm (A254) were highest for most terrestrial humic-like components, followed by the microbial humic-like component, and the protein-like components. Differences in size-resolved fluorescence were observed between DOM in humic-rich tributaries and in the mainstem of the LAR upstream of tributary inputs. The trend of variations in the A254-normalized PARAFAC loadings of terrestrial humic-like components also illustrates conservative mixing of aromatic-rich terrestrial DOM across size fractions in the LAR. From a molecular point of view, the mixing of fluorescent DOM occurred linearly and simultaneously across sizes without any evidence of aggregation, sedimentation, or changes in the fluorescence or concentration of any size fraction over the >60 km required for complete mixing of the river and its tributaries. Overall, this study provides insights into the size characteristics of fluorescent components of DOM and their conservative mixing behavior in large boreal rivers.

Asymmetrical flow field-flow fractionation combined with liquid chromatography enables rapid, quantitative, and structurally informative detection of resistant starch

Resistant starch (RS) is one kind of biomacromolecules with prebiotic effects. Nowadays, it is still a challenge to characterize structure and content of RS over entire molecular weight (Mw) distribution. In this study, a combined technique of asymmetrical flow field-flow fractionation (AF4) and liquid chromatography (LC) for the accurate structure characterization and quantitative analysis of RS was developed. AF4 coupled online with multiangle light scatting (MALS) and differential refractive index (dRI) detectors (AF4-MALS-dRI) was used to characterize the structural information (radius of gyration and Mw) and content of RS with Mw larger than 5 kDa. Meanwhile, RS was characterized by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The glucose content in the digested starch solution was quantitatively determined using LC. The results demonstrated that AF4-LC is a simple and rapid method for structurally informative detection and quantitative analysis of RS over entire Mw distribution.

The Contribution of Endmembers to Mixtures of Leaf Leachates and Riverine DOM can be Determined by Measuring Their Size and Fluorescence Properties

The molecular mass distribution (MMD) and fluorescence properties of dissolved organic matter (DOM) are important characteristics for tracing and predicting its pathways, processes, and fate in aquatic systems. For the first time, asymmetrical flow field-flow fractionation (AF4) with coupled absorbance and fluorescence detectors was used to determine the contribution of endmembers to three mixtures of leaf leachate and riverine DOM in various proportions. Parallel factor analysis (PARAFAC) and fractogram deconvolution were used to decompose and distinguish the size distributions and fluorescence excitation-emission matrices (EEMs) of mixture constituents. It was determined that: 1) Both size and optical properties were conservative tracers in mixtures; 2) Fractogram deconvolution was extremely helpful for discriminating endmember size properties; 3) The contributions of endmembers to overall DOC concentration were accurately estimated using both the proportion of a humic-like PARAFAC component (0.93 < R2 < 1.00), and the ratios of deconvoluted peaks (0.88 < R2 < 0.98). The fluorescence at the peak maximum of the MMD was lacking in protein-/polyphenol-like and microbial humic-like fluorescence compared to the whole sample (−11 ± 9 and −10 ± 7%, respectively); however, the contribution of endmembers to the MMD (A254) were also effectively predicted using both the proportion of a microbial humic-like PARAFAC component (0.91 < R2 < 0.98) and the ratio of deconvoluted peaks (0.94 < R2 < 0.98).

Cascade Filtration With PCR Detection and Field-Flow-Fractionation Online With ICP-MS for the Characterization of DNA Interaction With Suspended Particulate Matter.

The variety of applied antibiotics in animal and human medicine results in the release, development, and spread of relevant numbers of antibiotic resistance genes (ARGs) in the environment. The majority of ARGs are present in intracellular forms (in bacteria). Neglected aspects are extracellular variants of ARGs (eARGs) and their fragments, which have been detected in surface-water samples and sediments. The stability of eARGs is expected to be low; however, binding to particulate matter is likely to improve their stability and also affect their transport and dissemination behavior. Few studies have investigated DNA particle interactions, mostly via indirect characterization of adduct formation in model systems but not in real environmental matrices. Therefore, our study aims at a novel approach for direct characterization of desoxyribonucleic acid (DNA) particle interactions using both cascade filtration and field-flow fractionation. Cascade filtration with quantitative polymerase chain reaction (qPCR) detection indicated retention of ARGs on filters with much larger pore sizes supporting the hypothesis of ARG-particle interactions. However, artifacts from membrane clogging or DNA–membrane interaction cannot be excluded. Consequently, asymmetric flow field-flow fractionation was investigated as an alternative separation technique with the advantage of particle separation in a thin channel, reducing the risk of artifacts. The key method parameters, membrane composition, molecular weight cut off, and carrier composition, were systematically investigated using a calf-thymus DNA-spiked surface-water sample as a model. The results clearly showed a shift in the elution time of clay particles suggesting the presence of DNA–clay adducts. Multi-element detection by inductively coupled plasma mass spectrometry (ICP-MS) enabled monitoring of clay via the Al, Fe, and Si signals and DNA via the P signal. Matching peak profiles for the new fraction in the fractograms of the ARG and DNA-spiked water sample support adduct formation. Further evidence was provided by a novel post-channel filtration approach for the separation of free DNA from DNA–clay adducts.

Enzymatic modification of oat globulin enables covalent interaction with procyanidin B2

The effect of enzyme treatment on protein–tannin interactions was investigated using up-to-date analytical approaches for improving their physical properties. The formation of ligands between procyanidin B2 and native oat globulin (OG) was observed to be affected by the ratio of procyanidin B2 to OG and the availability of tryptophan. For the transglutaminase-treated OG, the results obtained from circular dichroism (CD) and size exclusion chromatography (SEC) revealed that procyanidin B2 acted as an acyl acceptor in the process of OG deamidation. Procyanidin B2 also inhibited the non-covalent protein–protein interactions occurring between the aromatic side-chains or sedimentation of tryptophan aggregates. For trypsin-treated OG, procyanidin B2 interacted with phenylalanine and the tryptophan side-chain of OG. The inhibition of procyanidin B2 towards protein–protein aggregation was proved by the observation of CD, SEC and asymmetric flow field-flow fractionation.

Squaric Ester-Based Nanogels Induce No Distinct Protein Corona but Entrap Plasma Proteins into their Porous Hydrogel Network.

After intravenous administration of nanocarriers, plasma proteins may rapidly adsorb onto their surfaces. This process hampers the prediction of the nanocarriers' pharmacokinetics as it determines their physiological identity in a complex biological environment. Toward clinical translation it is therefore an essential prerequisite to investigate the nanocarriers' interaction with plasma proteins. Here, this work evaluates a highly "PEGylated" squaric ester-based nanogel with inherent prolonged blood circulation properties. After incubation with human blood plasma, the nanogels are isolated by asymmetrical flow-field flow fractionation. Multiangle light scattering measurements confirm the absence of significant size increases as well as aggregation upon plasma incubation. However, proteomic analyses by gel electrophoresis find minor absolute amounts of proteins (3 wt%), whereas label-free liquid chromatography mass spectrometry identify 65 enriched proteins. Interestingly, the relative abundance of these proteins is almost similar to their proportion in pure native plasma. Due to the nanogels' hydrated and porous network morphology, it is concluded that the detected proteins rather result from passive diffusion into the nanogel network than from specific interactions at the plasma particle interface. Consequently, these results do not indicate a classical surface protein corona but rather reflect the highly outer and inner stealth-like behavior of the porous hydrogel network.

Effect of the ABCA1 agonist CS-6253 on amyloid-β and lipoprotein metabolism in cynomolgus monkeys

BackgroundInducing brain ATP-binding cassette 1 (ABCA1) activity in Alzheimer’s disease (AD) mouse models is associated with improvement in AD pathology. The purpose of this study was to investigate the effects of the ABCA1 agonist peptide CS-6253 on amyloid-β peptides (Aβ) and lipoproteins in plasma and cerebrospinal fluid (CSF) of cynomolgus monkeys, a species with amyloid and lipoprotein metabolism similar to humans.MethodsCS-6253 peptide was injected intravenously into cynomolgus monkeys at various doses in three different studies. Plasma and CSF samples were collected at several time points before and after treatment. Levels of cholesterol, triglyceride (TG), lipoprotein particles, apolipoproteins, and Aβ were measured using ELISA, ion-mobility analysis, and asymmetric-flow field-flow fractionation (AF4). The relationship between the change in levels of these biomarkers was analyzed using multiple linear regression models and linear mixed-effects models.ResultsFollowing CS-6253 intravenous injection, within minutes, small plasma high-density lipoprotein (HDL) particles were increased. In two independent experiments, plasma TG, apolipoprotein E (apoE), and Aβ42/40 ratio were transiently increased following CS-6253 intravenous injection. This change was associated with a non-significant decrease in CSF Aβ42. Both plasma total cholesterol and HDL-cholesterol levels were reduced following treatment. AF4 fractionation revealed that CS-6253 treatment displaced apoE from HDL to intermediate-density- and low density-lipoprotein (IDL/LDL)-sized particles in plasma. In contrast to plasma, CS-6253 had no effect on the assessed CSF apolipoproteins or lipids.ConclusionsTreatment with the ABCA1 agonist CS-6253 appears to favor Aβ clearance from the brain.

Renewable energy powered membrane technology: Implications of adhesive interaction between membrane and organic matter on spontaneous osmotic backwash cleaning

Organic matter (OM) in surface and ground waters may cause membrane fouling that is laborious to clean once established. Spontaneous osmotic backwash (OB) induced by solar irradiance fluctuation has been demonstrated for early mineral scaling/organic fouling control in decentralised small-scale photovoltaic powered-nanofiltration/reverse osmosis (PV-NF/RO) membrane systems. However, various OM types will interact differently with membranes which in turn affects the effectiveness of OB. This work evaluates the suitability of spontaneous OB cleaning for eleven OM types (covering low-molecular-weight organics (LMWO), humic substances, polyphenolic compounds and biopolymers) regarding adhesive interactions with NF/RO membranes. The adhesive interactions were quantified by an asymmetric flow field-flow fractionation coupled with an organic carbon detector (FFFF-OCD). The underlying mechanism of OM-membrane adhesive interactions affecting OB cleaning was elucidated. The results indicate that humic acid (a typical humic substance) and tannic acid (a typical polyphenolic compound) induced stronger adhesive interaction with NF/RO membranes than biopolymers and LMWO. When the mass loss of an OM due to adhesion was below a critical range, the spontaneous OB is most effective (>85% flux recovery); and above this range, the OB becomes ineffective (<50% flux recovery). Polyphenolic compounds and humic substances resulted in lower OB cleaning efficiency, due to their higher aromatic content, enhancing hydrophobic interactions and hydrogen bonding. Calcium-facilitated adhesion of some OM types (such as humic substances, polyphenolics and biopolymers) increased irreversible organic fouling potential and weakened OB cleaning, which was verified by both FFFF-OCD and membrane filtration results. This work provides a guidance to formulate strategies to enhance spontaneous OB cleaning, such as first identifying the adhesion of OM in feedwater (surface and ground waters) using FFFF-OCD, and then removing “sticky” OM using suitable pre-treatment processes.

Exomeres and Supermeres: monolithic or diverse?

Extracellular vesicles (EVs), including exosomes and microvesicles, are far from being the only RNA-containing extracellular particles (EPs). Recently, new 35 nm-sized EPs were discovered by asymmetric-flow field-flow fractionation and termed "exomeres". Purification of exomeres was later performed by differential ultracentrifugation as well. More recently, the supernatant of the high-speed ultracentrifugation used to collect exomeres was further centrifuged to collect a new class of EP, termed "supermeres". Supermeres contain high quantities of extracellular RNA and are enriched in miR-1246. They are also replete in disease biomarkers and can induce metabolic and adaptive changes in recipient cells. Here, we reanalyzed proteomic and transcriptomic data obtained in this exciting study to obtain further insights into the molecular composition of exomeres and supermeres. We found that the top-ranking RNAs in supermeres correspond to the footprints of extracellular protein complexes. These complexes protect fragments of the small nuclear RNA U2 and the 28S rRNA from extracellular ribonucleases (exRNases). We suggest that intracellular nanoparticles such as the U2 ribonucleoprotein, ribosomes and LGALS3BP ring-like decamers are released into the extracellular space. These heterogeneous EPs might be further processed by exRNases and co-isolate by ultracentrifugation with other components of exomeres and supermeres. We look forward to continuing progress in defining exRNA carriers, bridging process definitions with molecular composition and function.

The characterization of molecular weight distribution and aggregation by asymmetrical flow field-flow fractionation of unmodified and oxidized alginate

After oxidation, the presence of aldehydes on alginate chains allows reaction with, making oxidized alginate (OA) attractive for selective functionalization and gelation (i.e. covalent-crosslinks). The molecular weight (MW) of OA is significantly reduced after oxidation. As MW determines mechanical properties and degradation of OA-based hydrogels, it is essential to precisely measure the MW of OAs as function of its degree of oxidation (DO). Size exclusion chromatography is used for the analysis of polymers, but it has limited ability to measure the distribution of polymers with high MW, without recognizing the presence of aggregates. This work describes the use of asymmetrical flow field-flow fractionation (AF4) coupled with refractive index and multi-angle light scattering detectors as an analytical method for the quantification of the MW of OAs. OAs with low DO (5–10%) had a weight average molecular weight (Mw) in the range of 35–67 kDa, with aldehydes concentration of 1–1.8 mmol/g. OAs with higher DO (35–50%) had lower Mw (20 kDa), with aldehyde concentration of 4.3–6.5 mmol/g. This is the first study reporting the comprehensive characterization of OAs using the precision of AF4 method to quantify their MW, and will contribute substantially to the formulation of OA-based hydrogels towards more advanced biomedical applications.

Raman spectroscopy combined with comprehensive gas chromatography for label-free characterization of plasma-derived extracellular vesicle subpopulations

Raman spectroscopy together with comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry (GCxGC–TOFMS) was employed to characterize exomere- (<50 nm) and exosome-sized (50–80 nm) EVs isolated from human plasma by the novel on-line immunoaffinity chromatography – asymmetric flow field-flow fractionation method. CD9+, CD63+, and CD81+ EVs were selected to represent general EV subpopulations secreted into plasma, while CD61+ EVs represented the specific EV subset derived from platelets. Raman spectroscopy could distinguish EVs from non-EV particles, including apolipoprotein B-100-containing lipoproteins, signifying its potential in EV purity assessment. Moreover, platelet-derived (CD61+) EVs of both exomere and exosome sizes were discriminated from other EV subpopulations due to different biochemical compositions. Further investigations demonstrated composition differences between exomere- and exosome-sized EVs, confirming the applicability of Raman spectroscopy in distinguishing EVs, not only from different origins but also sizes. In addition, fatty acids that act as building blocks for lipids and membranes in EVs were studied by GCxGC—TOF-MS. The results achieved highlighted differences in EV fatty acid compositions in both esterified (membrane lipids) and non-esterified (free fatty acids) fractions, indicating possible differences in membrane structures, biological functions, and roles in cell-to-cell communications of EV subpopulations.

Study on effects of preparation method on the structure and antioxidant activity of protein-Tremella fuciformis polysaccharide complexes by asymmetrical flow field-flow fractionation

Oxidation is an essential biological process for human life. In this study, low density lipoprotein-Tremella fuciformis polysaccharide (LDL-TFP) complexes were prepared by electrostatic and covalent methods. The effects of preparation method on the structure and antioxidant activity of LDL-TFP complexes were investigated by asymmetrical flow field-flow fractionation (AF4) coupled with ultraviolet–visible (UV/Vis), multiangle light scattering (MALS), and differential refractive index (dRI) detectors. The results showed that the electrostatic LDL-TFP complexes had a spherical structure, while the covalent LDL-TFP complexes had a rod-like structure as indicated by the ratio of Rg (radius of gyration) to Rh (hydrodynamic radius). Moreover, the results revealed that the antioxidant activity of the LDL-TFP complexes on the HepG2 could be related to the structure of LDL-TFP complexes. The antioxidant activity of LDL-TFP complexes formed by LDL modified with phospholipase A2 was further enhanced. This study would help expand the application of TFP.

Kinetics and interaction studies of anti-tetraspanin antibodies and ICAM-1 with extracellular vesicle subpopulations using continuous flow quartz crystal microbalance biosensor

Continuous flow quartz crystal microbalance (QCM) was utilized to study binding kinetics between EV subpopulations (exomere- and exosome-sized EVs) and four affinity ligands: monoclonal antibodies against tetraspanins (anti-CD9, anti-CD63, and anti-CD81) and recombinant intercellular adhesion molecule-1 (ICAM-1) or CD54 protein). High purity CD9+, CD63+, and CD81+ EV subpopulations of <50 nm exomeres and 50–80 nm exosomes were isolated and fractionated using our recently developed on-line coupled immunoaffinity chromatography – asymmetric flow field-flow fractionation system. Adaptive Interaction Distribution Algorithm (AIDA), specifically designed for the analysis of complex biological interactions, was used with a four-step procedure for reliable estimation of the degree of heterogeneity in rate constant distributions. Interactions between exomere-sized EVs and anti-tetraspanin antibodies demonstrated two interaction sites with comparable binding kinetics and estimated dissociation constants Kd ranging from nM to fM. Exomeres exhibited slightly higher affinity compared to exosomes. The highest affinity with anti-tetraspanin antibodies was achieved with CD63+ EVs. The interaction of EV subpopulations with ICAM-1 involved in cell internalization of EVs was also investigated. EV – ICAM-1 interaction was also of high affinity (nM to pM range) with overall lower affinity compared to the interactions of anti-tetraspanin antibodies and EVs. Our findings proved that QCM is a valuable label-free tool for kinetic studies with limited sample concentration, and that advanced algorithms, such as AIDA, are crucial for proper determination of kinetic heterogeneity. To the best of our knowledge, this is the first kinetic study on the interaction between plasma-derived EV subpopulations and anti-tetraspanin antibodies and ICAM-1.

Asymmetrical Flow Field-Flow Fractionation Coupled to ICP-MS for Characterization of Trace Metal Species in the Environment from Macromolecular to Nano-Assemblage Forms: Current Challenges for Quantification

Asymmetrical flow field-flow fractionation (AF4) is a powerful technique employed for the separation of macromolecules, nanoparticles, and their assemblages according to their hydrodynamic behavior. It is well known that at this size range, complex interactions can occur between components (e.g. surface adsorption, aggregation) controlling the fate of trace metals (TMs) bound to them. AF4 coupling to inductively coupled plasma mass spectrometry (ICP-MS) allows the quantification of metal-containing species at trace levels present in environmental and biological systems on a size-composition basis. The combination of AF4-ICP-MS with other online detectors provides additional information that allows the assessment of the origin of analytes present in mixtures and complex matrixes with minimal sample preparation, which is crucial for understanding the behavior of trace metal contaminants. Despite the increasing use of AF4-ICP-MS in environmental contexts, we acknowledge that the quantification of inorganic species using such combined techniques requires further development of standardized procedures and need certified reference materials. In this review, we also discuss critical endpoints within the ICP-MS instrument coupled to AF4 that need to be controlled before quantitative measurements can be validated. Then, we illustrate how the combination of different online detectors in addition to ICP-MS offers an integrated picture of natural components states, thus providing key information on the changes in behavior of trace metal species and metallic nanoparticles (MNPs) as observed in both environmental samples and biofluids.

Native RNA Purification Method for Small RNA Molecules Based on Asymmetrical Flow Field-Flow Fractionation.

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules.

FFF-based high-throughput sequence shortlisting to support the development of aptamer-based analytical strategies

Aptamers are biomimetic receptors that are increasingly exploited for the development of optical and electrochemical aptasensors. They are selected in vitro by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, but although they are promising recognition elements, for their reliable applicability for analytical purposes, one cannot ignore sample components that cause matrix effects. This particularly applies when different SELEX-selected aptamers and related truncated sequences are available for a certain target, and the choice of the aptamer should be driven by the specific downstream application. In this context, the present work aimed at investigating the potentialities of asymmetrical flow field-flow fractionation (AF4) with UV detection for the development of a screening method of a large number of anti-lysozyme aptamers towards lysozyme, including randomized sequences and an interfering agent (serum albumin). The possibility to work in native conditions and selectively monitor the evolution of untagged aptamer signal as a result of aptamer-protein binding makes the devised method effective as a strategy for shortlisting the most promising aptamers both in terms of affinity and in terms of selectivity, to support subsequent development of aptamer-based analytical devices.Graphical abstract

Asymmetrical Flow Field-Flow Fractionation Methods for Quantitative Determination and Size Characterization of Thiols and for Mercury Size Speciation Analysis in Organic Matter-Rich Natural Waters.

Asymmetrical flow field-flow fractionation (AF4) efficiently separates various macromolecules and nano-components of natural waters according to their hydrodynamic sizes. The online coupling of AF4 with fluorescence (Fluo) and UV absorbance (UV) detectors (FluoD and UVD, respectively) and inductively coupled plasma–mass spectrometry (ICP-MS) provides multidimensional information. This makes it a powerful tool to characterize and quantify the size distributions of organic and inorganic nano-sized components and their interaction with trace metals. In this study, we developed a method combining thiol labeling by monobromo(trimethylammonio)bimane bromide (qBBr) with AF4–FluoD to determine the size distribution and the quantities of thiols in the macromolecular dissolved organic matter (DOM) present in highly colored DOM-rich water sampled from Shuya River and Lake Onego, Russia. We found that the qBBr-labeled components of DOM (qB-DOM) were of humic type, characterized by a low hydrodynamic size (d h < 2 nm), and have concentrations <0.3 μM. After enrichment with mercury, the complexes formed between the nano-sized components and Hg were analyzed using AF4–ICP-MS. The elution profile of Hg followed the distribution of the UV-absorbing components of DOM, characterized by slightly higher sizes than qB-DOM. Only a small proportion of Hg was associated with the larger-sized components containing Fe and Mn, probably inorganic oxides that were identified in most of the samples from river to lake. The size distribution of the Hg–DOM complexes was enlarged when the concentration of added Hg increased (from 10 to 100 nM). This was explained by the presence of small iron oxides, overlapping the size distribution of Hg–DOM, on which Hg bound to a small proportion. In addition, to provide information on the dispersion of macromolecular thiols in colored DOM-rich natural water, our study also illustrated the potential of AF4–FluoD–UVD–ICP-MS to trace or quantify dynamic changes while Hg binds to the natural nano-colloidal components of surface water.

Immunochemical and biophysical characterization of inactivated Sabin poliovirus products: Insights into rapid quality assessment tools

The aim of this study was to demonstrate the strength of combining immunochemical and biophysical analysis tools for assessing the quality of Sabin inactivated poliovirus vaccine (Sabin-IPV) bulk products. We assessed Sabin-IPV serotypes 1, 2 and 3 from six different manufacturers and evaluated their comparability through biosensor analysis and biophysical characterization methods, including tryptophan fluorescence and asymmetrical flow field-flow fractionation – multi-angle light scattering analysis. These methods enabled us to assess antigenic as well as conformational and structural integrity profiles, respectively. Based on Sabin-IPV samples that were subjected to accelerated storage conditions, we revealed that existing immunochemical methods exhibit remarkably similar trends to the results obtained by the biophysical characterization methods. While the results underpin that the comparability of Sabin-IPV bulk products of different manufacturers is weak, information about their quality can rapidly be obtained by using both immunochemical and biophysical methods. Furthermore, the study highlights that quality assessment of Sabin-IPV can be obtained through biophysical techniques can complement the assessments performed with monoclonal antibodies and suggests that similar techniques could be employed to characterize other enteroviruses.

Inter-laboratory analysis of cereal beta-glucan extracts of nutritional importance: An evaluation of different methods for determining weight-average molecular weight and molecular weight distribution

In an interlaboratory study we compare different methods to determine the weight-average molecular weight (Mw) and molecular weight distribution of six cereal beta-glucan isolates of nutritional importance. Size-exclusion chromatography (SEC) with multi-angle light scattering (MALS), capillary viscometry, sedimentation velocity analytical ultracentrifugation and one asymmetric flow field-flow fractionation (AF4)-MALS method all yielded similar Mw values for mostly individual chains of dissolved beta-glucan molecules. SEC with post-column calcofluor detection underestimated the Mw of beta-glucan >500 × 103 g/mol. The beta-glucan molecules analysed by these methods were primarily in a random coil conformation as evidenced from individual Mark-Houwink-Kuhn-Sakurada (MHKS) scaling coefficients between 0.5 and 0.6 and Wales-Van Holde ratios between 1.4 and 1.7. In contrast, a second AF4-MALS method yielded much larger Mw values for these same samples indicating the presence and detection of beta-glucan aggregates. Storage of the six beta-glucan solutions in the dark at 4 °C for 4 years revealed them to be stable. This suggests an absence of storage-induced irreversible aggregation phenomena or chain-scission. Shear forces in SEC and the viscometer capillary and hydrostatic pressure in analytical ultracentrifugation probably led to the reversable dissociation of beta-glucan aggregates into molecularly dissolved species. Thus, all these methods yield true weight-average molecular weight values not biased by the presence of aggregates as was the case in one of the AF4 based methods employed.

Characterization on the impact of different clarifiers on the white wine colloids using Asymmetrical Flow Field-Flow Fractionation

Clarifiers are substances used during the winemaking process to enhance clarity and stability in the wines. The different clarifiers may alter removal capacities differently. In this study, the removal efficiency of seven common fining agents, divided into three groups (mineral clarifiers, synthetic polymeric clarifiers, and vegetable protein clarifiers), was analyzed with Asymmetrical Flow Field-Flow fractionation (AF4). Besides, the relationship between the removal capacity and different molecular and macromolecular properties has been evaluated. The results showed extensive removal of colloidal and macromolecular matter by the bentonites with potential impact on characteristic properties of the wine. The vegetable clarifiers showed a more profiled reduction, potentially preserving characteristics of the wine. The synthetic polymers showed a more limited removal efficiency but with a high affinity to remove colloidal phenols. The use of AF4-UV-MALS-dRI allowed the characterization of the wines after different clarification treatments, showing to be an analytical technique with a potential impact on the wine industry.