Asymmetrical Flow Field-flow Fractionation Research Articles

Smart Poly(Acrylic Acid)/Poly(Acrylamide) Microgels with Interpenetrating Polymer Network Structure

Microgels with precisely tuned properties are of great importance as drug delivery systems. Here, we report the synthesis of microgel particles (MGs) with an interpenetrating polymer network structure composed of poly(acrylic acid) (PAA) and polyacrylamide (PAAM) for their potential application as cationic drug carriers. The MG properties were investigated via several analytical techniques, such as Dynamic Light Scattering (DLS) and zeta potential (ZP) measurements, Diffusion Nuclear Magnetic Resonance (NMR) spectroscopy, Asymmetrical Flow Field-Flow Fractionation (AF4) and Transmission Electron Microscopy (TEM). The MGs show pH-dependent swelling behavior with a radius of ~100 nm at collapsed state (pH < 4.5) and swell up to ~450 nm (pH~7), while their ZP decreases from −5 to −40 mV, depending on their composition. The results of the conducted studies demonstrate the potential of synthesized microgels for drug delivery in the gastrointestinal tract.

Asymmetric flow field-flow fractionation for comprehensive characterization of hetero-aggregates made of nano-silver and extracellular polymeric substances

The present study explores the capability of asymmetrical flow field-flow fractionation (AF4) coupled online with diode array (DAD), fluorescence detectors (FLD), multi-angle light scattering (MALS) and dynamic light scattering (DLS) to characterize silver nanoparticles (nAg) hetero-aggregates formed with diatoms derived extracellular polymeric substances (EPS). The content of EPS varied from 10.5 to 105 mgC L-1 and nAg were dispersed at 4 mg L-1 in a freshwater medium. Good recoveries (∼ 76.9 ± 8.4 %) of nAg-EPS were obtained from AF4-DAD signals, but an anomalous elution was observed as EPS concentration increased: AF4 retention times decreased despite average gyration radii measured by MALS for nAg-EPS increased (from 16 nm to 24 nm), which suggests a change in the aggregation state, as evaluated by UV–Vis scans obtained from DAD. A regular Brownian relaxation of nAg-EPS was proven for each EPS concentration using these 2 detectors. The comparison of on-line and batch DLS measurements validated in addition, that no (dis)aggregation occurs upon injections. After a thorough comparison with classical AF4 using standards, the frit-inlet-AF4 was used. Slightly higher recovery (79.6 ± 4.6 %) was obtained but similar deviation of nAg-EPS elution occurred, excluding the implication of membrane differential fouling of nAg-EPS /conditioning effects of EPS. The investigation of physico-chemical parameters controlling the Brownian relaxation of nAg-EPS suggests the influence of nAg-EPS structure and EPS loading. This study demonstrates the suitable use of AF4 coupled to multiple detectors to probe-out eco-corona formation and characterize polydisperse systems containing NPs, EPS and their hetero-aggregates in freshwaters, even under a non-ideal size-fractionation scenario.

Evaluation of size-based distribution of components in VYXEOS® liposomal formulation using asymmetric flow field-flow fractionation

VYXEOS® is the first FDA-approved dual-API liposomal formulation containing two different chemotherapeutics, daunorubicin and cytarabine at a 1:5 molar ratio. Analysis of bulk formulation does not provide insight to size-based distribution of APIs and excipients, therefore asymmetrical flow field-flow fractionation (AF4) was utilized for the size-based separation of VYXEOS® liposomes and collected size fractions were further analyzed for the concentrations of APIs, lipid excipients, and copper. Analysis results revealed a significant variation in API distribution across the size fractions, with the larger liposomes encapsulating a higher ratio of cytarabine to daunorubicin compared to the smaller liposomes, while lipid excipient composition was held constant across the size range. We attribute the size-based variation of API ratios to structural change during API loading and sequence of API loading. Cytarabine is first passively loaded into liposomes containing copper gluconate, and then daunorubicin is actively loaded using copper gradient where the daunorubicin is retained in the liposomes as a copper complex. This method can be extended to characterize various single and dual-API liposomal nanocarriers during drug product development and/or post market quality control.

Lipid nanoparticle properties explored using online asymmetric flow field-flow fractionation coupled with small angle X-ray scattering: Beyond average characterisation

This study employs asymmetric flow field-flow fractionation online coupled with small angle X-ray scattering at a synchrotron beamline, along with multiple downstream detectors, including multi-angle light scattering, dual wavelength UV and dRI. This setup enables size-resolved characterization of lipid nanoparticles, allowing for a detailed comparison between empty and cargo-loaded lipid nanoparticles intended for nucleic acid delivery. Batch-mode characterization techniques, including cryogenic transmission electron microscopy and dynamic light scattering, alongside collection of fractions for offline characterization with liquid chromatography-charged aerosol detection, allowed for determination of the particle morphology, hydrodynamic radius, and the lipid composition over the size distribution. Cargo-containing and empty lipid nanoparticles show differences in density, and loaded particles exhibit a broader size distribution and a higher frequency of blebs at the surface. Both samples consist of spherical core–shell structured particles, with no distinguishable internal structure. A pivotal finding, often assumed until now, is that the mole fraction of each individual lipid component closely matches the original formulation. This work contributes to a more detailed understanding of lipid nanoparticles, supporting their continued development and rational design in medical applications.

Evaluation of amplicons by AF4 as assistant for deep comprehension of loop-mediated isothermal amplification combined with lateral flow assay

Loop-mediated isothermal amplification (LAMP) is a rapid and efficient method for DNA amplification, producing concatemers of varying lengths (amplicons). This study explores the characterization of LAMP amplicons using asymmetric flow field-flow fractionation (AF4) and their realization in LAMP – lateral flow assay (LFA) for point-of-care diagnostics. We examined LAMP products from the invA gene of Salmonella enterica using two specific primer sets and three methods: fluorescent staining with SYBR Green, electrophoretic detection, and AF4. LAMP products labeled with biotin and fluorescein (introduced through primers, 12 combinations) were detected by LFA due to binding the labels. The efficiency of amplicon detection by test strips varied depending on the primer combination: the color intensities for the best and worst combinations differed by more than 7 times. AF4 enabled precise detection of components from primers to amplicons up to 500 bp and revealed differences influenced by labeling and primer combinations. An important advantage of AF4, which was demonstrated for the first time, was the characterization of native complexes of amplicons with receptors. The analysis of amplicons in complexes with streptavidin, anti-fluorescein antibodies, or both receptors together using AF4 revealed significant differences in the fractograms. The shape and maxima of the absorption peaks at 260 nm depended on retention time in the range from 20 to 35 min, indicating the formation of complexes of different compositions. AF4 and LFA showed that smaller amplicons (200-300 bp) primarily contribute to the binding of detected labels, while longer amplicons (1000-1500 bp) do not enhance the signal due to structural complexities. Our findings demonstrate the potential of AF4 as a tool for deep comprehension of LAMP processes and the composition of the formed amplicon-receptor complexes, offering valuable insights for enhancing LAMP-LFA systems. This work provides a foundation for the advanced application of AF4 for nucleic acid amplification technologies.

AF4-UHPLC: Two-dimensional separation of macromolecules in four white wines from South-Western France

Proteins in wines contribute to “protein haze,” which, while not affecting health or taste, can detract from the visual appeal of wines to consumers. To mitigate this issue, winemakers commonly use fining agents such as bentonite, despite the high costs involved. To overcome these challenges, numerous studies employ various analytical methods to better understand the behaviour of proteins. A novel technique which separates compounds by size, Asymmetrical Flow-Field Flow Fractionation (AF4), allows for studying proteins without denaturing them. Given that most proteins share similar sizes, identification remains challenging. The aim of this work was initially to develop a new system enabling simultaneous analysis of the macromolecular profile of wines (proteins, mannoproteins) using AF4 and real-time protein nature analysis (hydrophobicity properties) using Ultra-High Performance Liquid Chromatography (UHPLC). By injecting two standards of different sizes and chemical nature, thaumatin protein and mannoproteins, the system was validated. The subsequent application of this system to Southwestern wines revealed distinct profiles across monovarietal white wines from four grape varieties. While the Colombard (COL) and Gros manseng (GM) varieties showed similar protein compositions with varying concentrations, the Len de l'el (LL) variety had only two types of protein and no protein was detected in the Mauzac (MZ) variety. Despite these variations, all varieties contained mannoproteins. This system shows promise for studying wine protein composition and could potentially find applications in other matrices.

Determination of particle number concentration for biological particles using AF4-MALS: Dependencies on light scattering model and refractive index

Determining accurate counts and size distributions for biological particles (bioparticles) is crucial in wide-ranging fields, but current methods to this end are susceptible to bias from polydispersity in size. This bias can be mitigated by incorporating a separation step prior to characterization. For this reason, asymmetrical flow field-flow fractionation (AF4) with on-line multiangle light scattering (MALS) has become an important platform for determining particle size. AF4-MALS has also been increasingly used to report particle concentration, particularly for complex biological particles, yet the impact of light scattering models and particle refractive indices (RI) have not been quantitatively evaluated. Here, we develop an analysis workflow using AF4-MALS to simultaneously separate and determine particles sizes and concentrations. The impacts of the MALS particle counting model used to process data and the chosen RI value(s) on particle counts are systematically assessed for polystyrene latex (PSL) particles and bacterial outer membrane vesicles (OMVs) in the 20–500 nm size range. Across spherical models, PSL and OMV particle counts varied up to 13 % or 200 %, respectively. For the coated-sphere model used in the analysis of OMV samples, the sphere RI value greatly impacts particle counts. As the sphere RI value approaches the RI of the suspending medium, the model becomes increasingly sensitive to the light scattering signal-to-noise ratio ultimately causing erroneous particle counts. Overall, this work establishes the importance of selecting appropriate MALS models and RI values for bioparticles to obtain accurate counts and provides an AF4-MALS method to separate, enumerate, and size polydisperse bioparticles.

A clearer understanding of the dynamic structuring of different natural rubber genotypes on a macroscopic and mesoscopic scale by asymmetrical-flow field-flow fractionation (A4F) analysis

Unlike synthetic elastomers, the structure of natural rubber (NR) evolves (dynamic structuring) and so do its properties during the storage before reaching an industrial mixer. In the rubber industry this is known as storage hardening. NR samples from three genotypes (GT1, RRIM600 and PB235) were subjected to different levels of structuring by varying the structuring time (t) on phosphorus pentoxide (0 < t < 28 h). Storage hardening (ΔP) of the samples was then determined by measuring the increase in Wallace plasticity (P) (macro-scale) and by analyzing their mesostructure (meso-scale) using asymmetrical flow field flow fractionation (A4F). Monitoring ΔP as a function of structuring time revealed a diversity of behaviors specific to the genotype from which the rubber originated. For example, NR samples from genotypes GT1 and PB235 exhibited different kinetics for t < 12 h, an increase in ΔP with structuring time, but reached the same final plateau (t > 12 h). An A4F analysis of the samples was used to quantify the fraction of microaggregates smaller than 1 μm (microgel<1μ). The microgel<1μ rate decreased with structuring time to varying extents depending on the genotype. A very significant negative relationship was found between ΔP and the microgel<1μ rate, indicating that the NR samples that hardened the most contained the lowest microgel<1μ rate, but the highest macrogel rate.

Aqueous and Colloidal Dynamics in Size-Fractionated Paddy Soil Aggregates with Multiple Metal Contaminants under Redox Alternations.

Soil contamination by multiple metals is a significant concern due to the interlinked mobilization processes. The challenges in comprehending this issue arise from the poorly characterized interaction among different metals and the complexities introduced by spatial and temporal heterogeneity in soil systems. We delved into these complexities by incubating size-fractionated paddy soils under both anaerobic and aerobic conditions, utilizing a combination of techniques for aqueous and colloidal analysis. The contaminated paddy soil predominantly consisted of particles measuring <53, 250-53, and 2000-250 μm, with the <53 μm fractions exhibiting the highest concentrations of multiple metals. Interestingly, despite their higher overall content, the <53 μm fractions released less dissolved metal. Furthermore, glucose enhanced the release of arsenic while simultaneously promoting the sequestration of other metals, such as Pb, Zn, and Cu. Utilizing asymmetric flow field-flow fractionation, we unveiled the presence of both fine (0.3-130 kDa) and large (130-450 nm) colloidal pools, each carrying various metals with different affinities for iron minerals and organic matter. Our results highlighted the pivotal role of the <53 μm fraction as a significant reservoir for multiple metal contaminants in paddy soils, in which the colloidal metals were mainly associated with organic matter. These findings illuminated the size-resolved dynamics of soil metal cycling and provided insights for developing remediation strategies for metal-contaminated soil ecosystems.

Assessment of the impact of pasteurisation on protein denaturation in sheep sweet whey by asymmetrical flow field-flow fractionation according to sampling period

Whey recovery is limited in artisanal cheese manufacture. This by-product of cheese manufacture was traditionally considered as waste and is highly polluting. However, interest in the nutritional properties of native whey and its proteins has recently increased. Pretreatment is required to preserve highly perishable whey for processing. Only a few studies have focused on sheep whey, despite its considerable nutritional and technological potential. Here, we investigated the effect of heat treatment on the denaturation and formation of protein aggregates in sweet sheep whey collected in January, April and July. Two pasteurisation protocols were studied (72 °C for 1 min and 80 °C for 15 s). Microbiological quality was assessed by checking for the presence of microbes after five days of storage at 4 °C. Asymmetrical flow field-flow fractionation (AF4) coupled to UV detector, multiangle light scattering (MALS) and refractometer (dRi) is a mild, non-naturing technique for determining the extent of denaturation of whey proteins and the size of whey protein aggregates. Greater whey protein denaturation and larger whey protein aggregates were observed for the 80 °C/15 s protocol than for the 72 °C/1 min protocol, for the January and April samples. A decrease in Immunoglobulin G (IgG) content was observed after heat treatments in the samples from July but not significantly for the other proteins. Moreover, the retention times of the monomeric whey protein peaks on the AF4 fractogram were higher for this period, indicating that the proteins were larger. Microbiological testing showed that both pasteurisation treatments were sufficiently effective to ensure good sanitary quality. The pasteurisation schedule best preserving native proteins was heating at 72 °C for 1 min.

Assessing the size transformation of nanoplastics in natural water matrices

Understanding the stability of NPs in different aqueous environments, related with their size is crucial for assessing their potential risks. This is influenced by several factors, including pH, ionic strength, and the presence of biomolecules, or dissolved organic matter (DOM). In this study, dispersions of NPs derived from common plastic waste materials, including polystyrene (PS), polyethylene terephthalate (PET), polyvinyl chloride (PVC), polymethyl methacrylate (PMMA), and polycarbonate (PC), were synthesized by a nanoprecipitation method with sizes: 189 ± 7, 58 ± 3, 123 ± 4, 151 ± 7 and 182 ± 6 nm, respectively. Stability for a period of 14 days of these NPs was assessed in various natural water matrices. Different analytical techniques were used, including Asymmetric Flow Field-Flow Fractionation (AF4) coupled with UV–Vis and Dynamic Light Scattering (DLS) in series, batch DLS, Fourier-Transform Infrared Spectroscopy-Attenuated Total Reflection (FTIR-ATR), and Transmission Electron Microscopy (TEM). None of the studied NPs was stable in seawater and NPs were transformed in microplastics (MPs) by aggregation. PET was more prone to aggregation in all waters and PS was the most stable followed for PC, PVC and PMMA. However, bottle and tap waters maintained better the original size of NPs. For the most stable dispersion PS, the influence of heteroaggregation in tap and lagoon waters and aging from exposure to UV light in sea water were tested. In both cases, the stability over time was worse for PS. The results can contribute to a more comprehensive understanding of the fate and behaviour of NPs in natural aquatic environments, emphasizing the importance of studying a wide range of polymers.

Insight into protein cross-linking and casein polymerization in pre- and post-hydrolyzed lactose-free UHT milk during long-term storage

During processing and storage of both conventional and lactose-hydrolyzed UHT milk (LHM), aggregation of milk proteins occurs. Protein aggregation can inter alia occur via non-reducible covalent cross-links derived from either Maillard or dehydroalanine (DHA) pathways. To study this further in relation to processing method and lactase enzyme purity, LHM was produced using 3 different lactase preparations, with lactase enzymes added in a dairy setting either before (pre-hydrolysis) or after (post-hydrolysis) UHT treatment. The prepared LHM types were subsequently stored at either 25°C or 35°C for up to one year. Mass spectrometry was used to absolutely quantify the level of furosine, N-ɛ-(carboxymethyl)lysine (CML) and N-ɛ-(carboxyethyl)lysine (CEL), lanthionine (LAN) and lysinoalanine (LAL) in these products using a newly developed method on Triple Q for these processing-induced markers. The results showed higher levels of Maillard related processing markers in pre-hydrolyzed LHM compared with post-hydrolyzed LHM and conventional UHT milk which, on the other hand, contained higher concentrations of DHA-derived cross-links. Proteomics of collected particles from asymmetrical flow field-flow fractionation (AsFlFFF) in combination with gel electrophoresis indicated presence of intra-micellar cross-links during storage, for especially larger particles involving αS1- and αS2-caseins.

Characterization of Nanoparticles in Drinking Water Using Field-Flow Fractionation Coupled with Multi-Angle Light Scattering and Inductively Coupled Plasma Mass Spectrometry

The current absence of well-established and standardized methods for characterizing submicrometer- and nano-sized particles in water samples presents a significant analytical challenge. With the increasing utilization of nanomaterials, the potential for unintended exposure escalates. The widespread and persistent pollution of water by micro- and nanoplastics globally is a concern that demands attention, not only to reduce pollution but also to develop methods for analyzing these pollutants. Additionally, the analysis of naturally occurring nano entities such as bubbles and colloidal matter poses challenges due to the lack of systematic and consistent methodologies. This study presents Asymmetric Flow Field-Flow Fractionation (AF4) separation coupled with a UV-VIS spectrometer followed by Multi-Angle Light Scattering (MALS) for detection and size characterization of nanometric entities. It is coupled with an Inductively Coupled Plasma Mass Spectrometer (ICP-MS) for elemental analysis. Water samples from different sources, such as untreated mountain spring water, groundwater, and bottled drinking water, were analyzed. The system was calibrated using pure particle standards of different metallic compositions. Our study demonstrates the capability of AF4-UV-MALS-ICP-MS to detect metals such as Al, Ba, Cu, and Zn in particles of around 200 nm diameter and Mg associated with very small particles between 1.5 and 10 nm in different drinking water samples.

Nanofiltration membranes in asymmetric flow field-flow fractionation for improved organic matter size fractionation

Size fractionation of organic matter (OM) by asymmetric flow field-flow fractionation (FFFF) with ultrafiltration (UF) is limited by solute loss and peak resolution, particularly for low molecular weight fractions (<10 kDa). Nanofiltration (NF) with a molecular weight cut-off (MWCO) of ≤1 kDa can achieve significant OM retention and may improve OM fractionation. NF membranes with MWCOs ranging from 0.3 to 3 kDa were used to evaluate the retention and fractionation of nine OMs in the humic, polyphenol, biopolymer, and low molecular weight organic classes in stirred cell NF and FFFF. Membranes with an MWCO of 0.2–0.3 kDa (with a pure water permeability of 5–16 L.m−2.h−1.bar−1) with low OM interaction exhibited high peak recovery and resolution and hence improved OM fractionation. Loose NF (MWCO 3 kDa) exhibited a poor peak recovery because of a high OM loss due to both adhesion and permeation, particularly at high ionic strengths (>10 mM). A mixture of nine types of OM and natural organic matter samples from rivers, swamps and lakes achieved good fractionation with a 0.3 kDa (NF270) membrane at low ionic strength. The performance of NF for the analysis of OM and colloids in the smallest size range (≤1 kDa MW) could be further improved by optimizing the salt composition of the mobile phase and by overcoming the pressure limitations of FFFF channels.

Development of an advanced separation and characterization platform for mRNA and lipid nanoparticles using multi-detector asymmetrical flow field-flow fractionation.

In recent years, the use of lipid nanoparticles (LNPs) for delivery of messenger RNA (mRNA)-based therapies has gained substantial attention in the field of drug development. In such an application, multiple LNP attributes have to be carefully characterized to ensure product safety and quality, whereas accurate and efficient characterization of these complex mRNA-LNP formulations remains a challenging endeavor. Here, we present the development and application of an online separation and characterization platform designed for the isolation and in-depth analysis of mRNAs and mRNA-loaded LNPs. Our asymmetrical flow field-flow fractionation with a multi-detector (MD-AF4) method has demonstrated exceptional resolution between mRNA-LNPs and mRNAs, delivering excellent recoveries (over 70%) for both analytes and exceptional repeatability. Notably, this platform allows for comprehensive and multi-attribute LNP characterization, including online particle sizing, morphology characterization, and determination of encapsulation efficiency, all within a single injection. Furthermore, real-time online sizing by synchronizing multi-angle light scattering (MALS) and dynamic light scattering (DLS) presented higher resolution over traditional batch-mode DLS, particularly in differentiating heterogeneous samples with a low abundance of large-sized particles. Additionally, our method proves to be a valuable tool for monitoring LNP stability under varying stress conditions. Our work introduces a robust and versatile analytical platform using MD-AF4 that not only efficiently provides multi-attribute characterizations of mRNA-LNPs but also holds promise in advancing studies related to formulation screening, quality control, and stability assessment in the evolving field of nanoparticle delivery systems for mRNAs.

Polyamines putrescine and spermidine as modulators of protein aggregation rate: The effect on DTT-induced aggregation of α-lactalbumin

Protein aggregation is undesirable for cells due to its possible toxicity, and is also undesirable in biotechnology and pharmaceuticals. Polyamines are known to be capable of both suppressing and stimulating protein aggregation. In the present work polyamines (spermidine, putrescine) have been shown to alter the pathway of α-lactalbumin aggregation induced by dithiothreitol, leading to the formation of larger protein particles during the initial stages of aggregation and promoting the later stage of sticking of aggregates. According to the aggregation kinetics data, polyamines accelerate protein aggregation in a concentration-dependent manner, with a maximum at 50 mM spermidine and 100 mM putrescine. With a further increase in polyamines concentration the effect of aggregation acceleration decreased, thus, the modulation of the aggregation rate by polyamines was shown. A comparison of the aggregation kinetics and hydrodynamic radii growth data registered by dynamic light scattering with the data obtained by asymmetric flow field-flow fractionation and analytical ultracentrifugation allowed us to describe the early stages of aggregation and formation of initial α-lactalbumin clusters. Our results provide a deeper insight into the mechanism of amorphous aggregation of α-lactalbumin and polyamines action on protein aggregation and protein-protein interaction in general.

Engineering hyaluronic acid-based nanoassemblies for monoclonal antibody delivery – design, characterization, and biological insights

AbstractThe current spotlight of cancer therapeutics is shifting towards personalized medicine with the widespread use of monoclonal antibodies (mAbs). Despite their increasing potential, mAbs have an intrinsic limitation related to their inability to cross cell membranes and reach intracellular targets. Nanotechnology offers promising solutions to overcome this limitation, however, formulation challenges remain. These challenges are the limited loading capacity (often insufficient to achieve clinical dosing), the complex formulation methods, and the insufficient characterization of mAb-loaded nanocarriers. Here, we present a new nanocarrier consisting of hyaluronic acid-based nanoassemblies (HANAs) specifically designed to entrap mAbs with a high efficiency and an outstanding loading capacity (50%, w/w). HANAs composed by an mAb, modified HA and phosphatidylcholine (PC) resulted in sizes of ~ 100 nm and neutral surface charge. Computational modeling identified the principal factors governing the high affinity of mAbs with the amphiphilic HA and PC. HANAs composition and structural configuration were analyzed using the orthogonal techniques cryogenic transmission electron microscopy (cryo-TEM), asymmetrical flow field-flow fractionation (AF4), and small-angle X-ray scattering (SAXS). These techniques provided evidence of the formation of core-shell nanostructures comprising an aqueous core surrounded by a bilayer consisting of phospholipids and amphiphilic HA. In vitro experiments in cancer cell lines and macrophages confirmed HANAs’ low toxicity and ability to transport mAbs to the intracellular space. The reproducibility of this assembling process at industrial-scale batch sizes and the long-term stability was assessed. In conclusion, these results underscore the suitability of HANAs technology to load and deliver biologicals, which holds promise for future clinical translation.

Optimized AF4 combined with density cushion ultracentrifugation enables profiling of high-purity human blood extracellular vesicles.

Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, the identification of EVs derived from blood samples is hindered by the presence of abundant plasma proteins, which impairs the downstream biochemical analysis of EV-associated proteins and nucleic acids. Here, we employed optimized asymmetric flow field-flow fractionation (AF4) combined with density cushion ultracentrifugation (UC) to obtain high-purity and intact EVs with very low lipoprotein contamination from human plasma and serum. Further proteomic analysis revealed more than 1000 EV-associated proteins, a large proportion of which has not been previously reported. Specifically, we found that cell-line-derived EV markers are incompatible with the identification of plasma-EVs and proposed that the proteins MYCT1, TSPAN14, MPIG6B and MYADM, as well as the traditional EV markers CD63 and CD147, are plasma-EV markers. Benefiting from the high-purity of EVs, we conducted comprehensive miRNA profiling of plasma EVs and nanosized particles (NPs), as well as compared plasma- and serum-derived EVs, which provides a valuable resource for the EV research community. Overall, our findings provide a comprehensive assessment of human blood EVs as a basis for clinical biopsy applications.

On-line coupling of hollow-fiber flow field-flow fractionation and depolarized multi-angle static light scattering (HF5/D-MALS). Proof of principle

Introduced here is the on-line coupling of hollow-fiber flow field-flow fractionation (HF5) to depolarized multi-angle static light scattering (D-MALS). HF5 is a size-based separation alternative to size-exclusion and hydrodynamic chromatography and asymmetric flow field-flow fractionation. HF5 can separate larger sizes than its chromatographic counterparts and provides several advantages over its fractionation counterpart, including reduced sample consumption and greater ease of operation. D-MALS is a variant of MALS in which the depolarized scattering from the analyte solution is measured at a variety of angles simultaneously. Measurements of depolarized scattering have previously been employed in studying the optical properties of solutions or suspensions, to determine the length of rod-like analytes, and to gain increased accuracy in the determination of analyte molar mass. The coupling HF5/D-MALS allows for the depolarization ratio of a solution or suspension to be measured continuously across the fractogram. This is demonstrated here for a Teflon latex the size range of which extends beyond that accessible to commercial size-exclusion columns. The results presented provide the first reported on-line HF5/D-MALS coupling, showing the feasibility of the technique as well as its realized potential for providing continuous depolarization measurements, inter alia.

Delicate Analysis of Interacting Proteins and Their Assemblies by Flow Field-Flow Fractionation Techniques.

We study the efficiency of several asymmetrical flow field-flow fractionation (AF4) techniques to investigate self-associating wheat gluten proteins. We compare the use of a denaturing buffer including sodium dodecyl sulfate (SDS) and a mild chaotropic solvent, water/ethanol, as the eluent, on a model gluten sample. Through a thorough analysis of the data obtained from coupled light scattering detectors and with the identification of molecular composition of the eluted protein, we evidence coelution events in several conditions. We show that the focus step used in conventional AF4 with the SDS buffer leads to the formation of aggregates that coelute with monomeric proteins. By contrast, a frit-inlet device enables the fractionation of individual wheat proteins in the SDS buffer. Interestingly conventional AF4, using water/ethanol as eluent, is an effective method for fractionating gluten proteins and their complex dynamic assemblies, which involve weak forces and are composed of both monomeric and polymeric proteins.