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.

Centrifugal Field-Flow Fractionation Enables Detection of Slight Aggregation of Nanoparticles That Impacts Their Biomedical Applications.

Nanoparticles (NPs) are anticipated to be used for various biomedical applications in which their aggregation has been an important issue. However, concerns regarding slightly aggregated but apparently monodispersed NPs have been difficult to address because of a lack of appropriate evaluation methods. Here, we report centrifugal field-flow fractionation (CF3) as a powerful method for analyzing the slight aggregation of NPs, using antibody-modified gold NPs (Ab-AuNPs) prepared by a conventional protocol with centrifugal purification as a model. While common evaluation methods such as dynamic light scattering cannot detect significant signs of aggregation, CF3 successfully detects distinct peaks of slightly aggregated NPs, including dimers and trimers. Their impact on biological interactions was also demonstrated by a cellular uptake study: slightly aggregated Ab-AuNPs exhibited 1.8 times higher cellular uptake than monodispersed Ab-AuNPs. These results suggest the importance of aggregate evaluation via CF3 as well as the need for careful attention to the bioconjugation procedures for NPs.

Comparison of a thickness-tapered channel in flow field-flow fractionation with a conventional channel with flow rate programming.

The thickness-tapered channel structure in flow field-flow fractionation (FlFFF), recently introduced by constructing a channel with a linear decrease in thickness along its length, demonstrated effectiveness in steric/hyperlayer separation of supramicron particles with improvements in separation speed, elution recovery, and an expanded dynamic size range of separation. In this study, we conducted a comparative analysis of the performance between the impact of field (or crossflow rate) programming or outflow rate programming for the separation of polystyrene latex standards (50 ∼ 800 nm) with a conventional channel having uniform thickness and a thickness-tapered channel without programming. Outlet flow rate and crossflow rate conditions were also varied. Although the particle size resolution of the tapered channel does not surpass that of field programming in uniform thickness channel, it achieves higher-speed separation without a significant loss of resolution and without the need for a complex flow controller system even at a low outflow rate condition. Furthermore, it yielded an improved resolution for particles close to the steric transition regime (400 ∼ 600 nm) in the normal mode of separation. Due to the continuous increase in mean flow velocity down the channel, the tapered channel exhibits flexibility in separating submicron-sized particles at high crossflow rate conditions or low outflow rate conditions, of which the latter can be advantageous when coupled with mass spectrometry in a miniaturized setup.

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 (by static light scattering) to the measured hydrodynamic radius (by dynamic 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).

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.

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

Arabinoxylans, ß-glucans, and dextrins influence the brewing industry's filtration process and product quality. Despite their relevance, only a maximum concentration of ß-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 ß-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.

Quantitative determination of the intracellular uptake of silica nanoparticles using asymmetric flow field flow fractionation coupled with ICP mass spectrometry and their cytotoxicity in HepG2 cells.

We established a size separation method for silica nanoparticles (SiNPs) measuring 10, 30, 50, 70, and 100 nm in diameter using asymmetric flow field flow fractionation hyphenated with inductively coupled plasma mass spectrometry (AF4-ICP-MS), and evaluated the cytotoxicity of SiNPs in human hepatoma HepG2 cells. Analysis of the mixture sample revealed that nanoparticles of different sizes were eluted at approximately 2-min intervals, with no effect on each elution time or percentage recovery. Compared with larger SiNPs, smaller SiNPs exhibited high cytotoxicity when the volume of SiNPs exposed to the cells was the same. We measured SiNPs in culture medium and inside cells by AF4-ICP-MS and found that approximately 17% of SiNPs in the mixture of five differently sized particles were absorbed by the cells. Transmission electron microscopy revealed that 10 nm SiNPs formed aggregates and accumulated in the cells. Based on AF4-ICP-MS analysis, there is no clear difference in the particle volume absorbed by the cells among different sizes. Therefore, the high toxicity of small SiNPs can be explained by the fact that their large surface area relative to particle volume efficiently induces toxicological influences. Indeed, the large surface area of 10 nm SiNPs significantly contributed to the production of reactive oxygen species.

Rapid and green discrimination of bovine milk according to fat content, thermal treatment, brand and manufacturer via colloidal fingerprinting.

Addressing food safety and detecting food fraud while fulfilling greenness requisites for analysis is a challenging but necessary task. The use of sustainable techniques, with limited pretreatment, non-toxic chemicals, high throughput results, is recommended. A combination of Field Flow Fractionation (FFF), working in saline carrier and with minimal preprocessing, and chemometrics was for the first time applied to bovine milk grouping. A set of 47 bovine milk samples was analyzed: a single analysis yielded a characteristic multidimensional colloidal dataset, that once processed with multivariate tools allowed simultaneously for different discriminations: fat content, thermal treatment, brand and manufacturing plant. The analytical methodology is fast, green, simple, and inexpensive and could offer great help in the field of quality control and frauds identification. This work represents also the first attempt to identify milk sub-typologies based on colloidal profiles, and the most complete study concerning multivariate analysis of FFF fingerprint.

Field-flow fractionation - an excellent tool for fractionation, isolation and/or purification of biomacromolecules.

Field-flow fractionation (FFF) with its several variants, has developed into a mature methodology. The scope of the FFF investigations has expanded, covering both a wide range of basic studies and especially a wide range of analytical applications. Special attention of this review is given to the achievements of FFF with reference to recent applications in the fractionation, isolation, and purification of biomacromolecules, and from which especially those of (in alphabetical order) bacteria, cells, extracellular vesicles, liposomes, lipoproteins, nucleic acids, and viruses and virus-like particles. In evaluating the major approaches and trends demonstrated since 2012, the most significant biomacromolecule applications are compiled in tables. It is also evident that asymmetrical flow field-flow fractionation is by far the most dominant technique in the studies. The industry has also shown current interest in FFF and adopted it in some sophisticated fields. FFF, in combination with appropriate detectors, handles biomacromolecules in open channel in a gentle way due to the lack of shear forces and unwanted interactions caused by the stationary phase present in chromatography. In addition, in isolation and purification of biomacromolecules quite high yields can be achieved under optimal conditions.

Sedimentation Field-Flow Fractionation: A Diagnostic Tool for Rapid Antimicrobial Susceptibility Testing.

Conventional antimicrobial susceptibility testing (AST) methods require 24-48 h to provide results, creating the need for a probabilistic antibiotic therapy that increases the risk of antibiotic resistance emergence. Consequently, the development of rapid AST methods has become a priority. Over the past decades, sedimentation field-flow fractionation (SdFFF) has demonstrated high sensitivity in early monitoring of induced biological events in eukaryotic cell populations. This proof-of-concept study aimed at investigating SdFFF for the rapid assessment of bacterial susceptibility to antibiotics. Three bacterial species were included (Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) with two panels of antibiotics tailored to each bacterial species. The results demonstrate that SdFFF, when used in "Hyperlayer" elution mode, enables monitoring of antibiotic-induced morphological changes. The percentage variation of the retention factor (PΔR) was used to quantify the biological effect of antibiotics on bacteria with the establishment of a threshold value of 16.8% to differentiate susceptible and resistant strains. The results obtained with SdFFF were compared to that of the AST reference method, and a categorical agreement of 100% was observed. Overall, this study demonstrates the potential of SdFFF as a rapid method for the determination of antibiotic susceptibility or resistance since it is able to provide results within a shorter time frame than that needed for conventional methods (3-4 h vs 16-24 h, respectively), enabling earlier targeted antibiotic therapy. Further research and validation are necessary to establish the effectiveness and reliability of SdFFF in clinical settings.

Increasing the Resolution of Field-Flow Fractionation with Increasing Crossflow Gradients.

The resolution of flow field-flow fractionation (flow FFF) depends primarily on the crossflow rate and its change over time. In this work, we demonstrate a method for modulation of the crossflow rate during separation that increases the peak-to-peak resolution of the resulting fractograms. In classical FFF methods, the crossflow rate is either maintained constant or decreased during the separation of the different species. In this work, higher resolution between peaks was achieved by a novel gradient method in which the crossflow is increased briefly during separation to allow stronger retention of the later eluting peaks. We first outline the theoretical basis by which improved separation is achieved. We confirm our hypothesis by quantifying the impact of increasing crossflow on the resolution between a monoclonal antibody monomer and its high-molecular-weight aggregate. We then demonstrate that this method is applicable to two different FFF methods (AF4 and HF5) and various pharmaceutically relevant samples (monoclonal antibodies and adeno-associated viruses). Finally, we hypothesize that increasing the force perpendicular to the laminar flow as described here is broadly applicable to all FFF methods and improves the quality of FFF-based separations.

Field-Flow Fractionation in Molecular Biology and Biotechnology.

Field-flow fractionation (FFF) is a family of single-phase separative techniques exploited to gently separate and characterize nano- and microsystems in suspension. These techniques cover an extremely wide dynamic range and are able to separate analytes in an interval between a few nm to 100 µm size-wise (over 15 orders of magnitude mass-wise). They are flexible in terms of mobile phase and can separate the analytes in native conditions, preserving their original structures/properties as much as possible. Molecular biology is the branch of biology that studies the molecular basis of biological activity, while biotechnology deals with the technological applications of biology. The areas where biotechnologies are required include industrial, agri-food, environmental, and pharmaceutical. Many species of biological interest belong to the operational range of FFF techniques, and their application to the analysis of such samples has steadily grown in the last 30 years. This work aims to summarize the main features, milestones, and results provided by the application of FFF in the field of molecular biology and biotechnology, with a focus on the years from 2000 to 2022. After a theoretical background overview of FFF and its methodologies, the results are reported based on the nature of the samples analyzed.

Proteomics protocol for obtaining extracellular vesicle from human plasma using asymmetrical flow field-flow fractionation technology.

Plasma extracellular vesicles (EVs) represent a potential resource for biomarkers of multiple diseases. Here, we present a protocol for obtaining EVs from human plasma using asymmetrical flow field-flow fractionation technology. We describe steps for using tandem mass tags to perform comparative proteomic studies of a large clinical cohort. We then detail targeted quantitative analysis of differential proteins based on a parallel reaction monitoring technique. For complete details on the use and execution of this protocol, please refer to Wu et al. (2020)1 and Li et al. (2023).2.

[Separation and characterization of Gastrodia elata polysaccharides based on asymmetrical flow field-flow fractionation: steric transition phenomenon].

Asymmetrical flow field-flow fractionation (AF4), a gentle tool for the separation and characterization of particles and macromolecules, has attracted increased interest in recent years owing to its broad dynamic size range and utilization of "open channel" voids in the packing or stationary phase. A steric transition phenomenon in which the sample elution mode change from the normal mode to the steric/hyperlayer mode occurs. Accurate characterization by AF4 requires the absence of steric transition, particularly when the sample has a broad size distribution, because the effect of the combination of different modes is difficult to interpret. In this study, the relative molecular mass (M), radius of gyration (Rg), and conformation of Gastrodia elata polysaccharides (GEPs) were characterized using AF4 coupled with online multi-angle light scattering (MALS) and differential refractive index (dRI) detection (AF4-MALS-dRI). Steric transition was observed during GEP separation by AF4 owing to the broad size distribution of the molecules. This phenomenon would result in the inaccurate characterization of the GEPs in terms of M and Rg because two GEP groups of different sizes may elute together. In this study, the effects of constant and exponentially decaying cross-flow rates, sample mass concentration, and spacer thickness on steric transition were systematically investigated. The results indicated that a high GEP mass concentration (i. e., 0.75 mg/mL) can lead to steric transition. The spacer thickness affected the resolution and retention time of the GEPs and changed the steric transition point (di). An exponentially decaying cross-flow rate not only adjusted the di of the polydisperse GEP samples but also improved the GEP resolution and shortened the analysis time. The influence of steric transition was solved under the following operating conditions: injected GEP mass concentration=0.5 mg/mL; injection volume=50 µL; spacer thickness=350 µm; detector flow rate=1.0 mL/min; and cross-flow rate exponentially decayed from 0.2 to 0.05 mL/min with a half-life of 2 min. Moreover, the influence of GEP origins and ultrasound treatment time on the M and Rg distributions and conformation of GEPs were investigated under the optimized operating conditions. The results showed that the M and Rg distributions of Yunnan and Sichuan GEPs decreased with increasing ultrasound time. When the ultrasound treatment time was 15 min, the Yunnan GEPs had a loosely hyperbranched chain conformation, whereas the Sichuan GEPs had a spherical conformation. When the ultrasound treatment time was increased to 30 or 60 min, the GEPs from both Yunnan and Sichuan had a hyperbranched chain conformation, indicating that ultrasound treatment resulted in GEP degradation. Under the same extraction conditions, GEPs from Yunnan had larger M and Rg values than those from Sichuan. AF4-MALS-dRI showed good repeatability for the characterization of GEPs under the optimized operating conditions. The relative standard deviations of Rg and M were 0.5% and 1.7%, respectively. The data presented in this study can be used as a starting point for in-depth studies on the structural bioactivity of GEPs.

Asymmetrical Flow Field Flow Fractionation for Molar Mass Characterization of Polyethylene Oxide in Abuse-Deterrent Formulations.

High molar mass polyethylene oxide (HM-PEO) is commonly used to enhance the mechanical strength of solid oral opioid drug products to deter abuse. Because the properties of PEO depend on molar mass distribution, accurately determining the molar mass distribution is a necessary part of understanding PEO's role in abuse-deterrent formulations (ADF). In this study, an asymmetrical flow field-flow fractionation (AF4) analytical procedure was developed to characterize PEO polymers with nominal molar masses of 1, 4 or 7 MDa as well as those from in-house prepared placebo ADF. The placebo ADF were manufactured using direct compress or hot-melt-extrusion methods, and subjected to physical manipulation, such as heating and grinding before measurement by AF4 were performed. The molar mass distribution characterized by AF4 revealed that PEO was sensitive to thermal stress, exhibiting decreased molar mass with increased heat exposure. The optimized AF4 method was deemed suitable for characterizing HM-PEO, offering adequate dynamic separation range for PEO with molar mass from 100 kDa to approximately 10 MDa.

Assessment of the Effects of Structural Modification of Gastrodia elata Polysaccharide on Anti-Breast Cancer Activity Using Asymmetrical Flow Field-Flow Fractionation.

Gastrodia elata ("Tian Ma" in Chinese) is used as a food and medical ingredient in traditional Chinese medicine. In this study, to enhance the anti-breast cancer activity of Gastrodia elata polysaccharide (GEP), GEPs were modified via sulfidation (SGEP) and acetylation (AcGEP). The physicochemical properties (such as solubility and substitution degree) and structural information (such as molecular weight Mw and radius of gyration Rg) of GEP derivatives were determined by Fourier transformed infrared (FTIR) spectroscopy and asymmetrical flow field-flow fractionation (AF4) coupled online with multiangle light scattering (MALS) and differential refractive index (dRI) detectors (AF4-MALS-dRI). The effects of the structural modification of GEP on the proliferation, apoptosis, and cell cycle of MCF-7 cell were studied systematically. The ability of MCF-7 cell for the uptake of GEP was studied by laser scanning confocal microscopy (LSCM). The results suggested that the solubility and anti-breast cancer activity of GEP were enhanced and the average Rg and Mw of GEP decreased after chemical modification. The AF4-MALS-dRI results showed that the chemical modification process simultaneously caused the degradation and aggregation of GEPs. The LSCM results revealed that more SGEP can enter the MCF-7 cell interior compared with AcGEP. The results indicated that the structure of AcGEP could play a dominating role in antitumor activity. The data obtained in this work can be used as a starting point for investigating the structure-bioactivity of GEPs.

Characterizing Non-covalent Protein Complexes Using Asymmetrical Flow Field-Flow Fractionation On-Line Coupled to Native Mass Spectrometry.

We report an online analytical platform based on the coupling of asymmetrical flow field-flow fractionation (AF4) and native mass spectrometry (nMS) in parallel with UV-absorbance, multi-angle light scattering (MALS), and differential-refractive-index (UV-MALS-dRI) detectors to elucidate labile higher-order structures (HOS) of protein biotherapeutics. The technical aspects of coupling AF4 with nMS and the UV-MALS-dRI multi-detection system are discussed. The "slot-outlet" technique was used to reduce sample dilution and split the AF4 effluent between the MS and UV-MALS-dRI detectors. The stability, HOS, and dissociation pathways of the tetrameric biotherapeutic enzyme (anticancer agent) l-asparaginase (ASNase) were studied. ASNase is a 140 kDa homo-tetramer, but the presence of intact octamers and degradation products with lower molecular weights was indicated by AF4-MALS/nMS. Exposing ASNase to 10 mM NaOH disturbed the equilibrium between the different non-covalent species and led to HOS dissociation. Correlation of the information obtained by AF4-MALS (liquid phase) and AF4-nMS (gas phase) revealed the formation of monomeric, tetrameric, and pentameric species. High-resolution MS revealed deamidation of the main intact tetramer upon exposure of ASNase to high pH (NaOH and ammonium bicarbonate). The particular information retrieved from ASNase with the developed platform in a single run demonstrates that the newly developed platform can be highly useful for aggregation and stability studies of protein biopharmaceuticals.

The Power of Field-Flow Fractionation in Characterization of Nanoparticles in Drug Delivery.

Asymmetric-flow field-flow fractionation (AF4) is a gentle, flexible, and powerful separation technique that is widely utilized for fractionating nanometer-sized analytes, which extend to many emerging nanocarriers for drug delivery, including lipid-, virus-, and polymer-based nanoparticles. To ascertain quality attributes and suitability of these nanostructures as drug delivery systems, including particle size distributions, shape, morphology, composition, and stability, it is imperative that comprehensive analytical tools be used to characterize the native properties of these nanoparticles. The capacity for AF4 to be readily coupled to multiple online detectors (MD-AF4) or non-destructively fractionated and analyzed offline make this technique broadly compatible with a multitude of characterization strategies, which can provide insight on size, mass, shape, dispersity, and many other critical quality attributes. This review will critically investigate MD-AF4 reports for characterizing nanoparticles in drug delivery, especially those reported in the last 10-15 years that characterize multiple attributes simultaneously downstream from fractionation.

Automated On-Line Isolation and Fractionation Method for Subpopulations of Extracellular Vesicles.

Immunoaffinity chromatography (IAC) with selective antibodies immobilized on polymeric monolithic disk columns enables selective isolation of biomacromolecules from human plasma, while asymmetrical flow field-flow fractionation (AsFlFFF or AF4) can be used for further fractionation of relevant subpopulations of biomacromolecules (e.g., small dense low-density lipoproteins, exomeres, and exosomes) from the isolates. Here we describe how the isolation and fractionation of subpopulations of extracellular vesicles can be achieved without the presence of lipoproteins using on-line coupled IAC-AsFlFFF. With the developed methodology, it is possible to have fast, reliable, and reproducible automated isolation and fractionation of challenging biomacromolecules from human plasma with a high purity and high yields of subpopulations.

Extracellular Vesicles Isolation from Large Volume Samples Using a Polydimethylsiloxane-Free Microfluidic Device.

Extracellular vesicles (EV) have many attributes important for biomedicine; however, current EV isolation methods require long multi-step protocols that generally involve bulky equipment that cannot be easily translated to clinics. Our aim was to design a new cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) asymmetric flow field fractionation microfluidic device that could isolate EV from high-volume samples in a simple and efficient manner. We tested the device with large volumes of urine and conditioned cell media samples, and compared it with the two most commonly used EV isolation methods. Our device was able to separate particles by size and buoyancy, and the attained size distribution was significantly smaller than other methods. This would allow for targeting EV size fractions of interest in the future. However, the results were sample dependent, with some samples showing significant improvement over the current EV separation methods. We present a novel design for a COC-OSTE microfluidic device, based on bifurcating asymmetric flow field-flow fractionation (A4F) technology, which is able to isolate EV from large volume samples in a simple, continuous-flow manner. Its potential to be mass-manufactured increases the chances of implementing EV isolation in a clinical or industry-friendly setting, which requires high repeatability and throughput.