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We describe the protocol development and optimization of asymmetric-flow field-flow fractionation (AF4) technology for separating and characterizing extracellular nanoparticles (ENPs), particularly small extracellular vesicles (sEVs), known as exosomes, and even smaller novel nanoparticles, known as exomeres. This technique fractionates ENPs on the basis of hydrodynamic size and demonstrates a unique capability to separate nanoparticles with sizes ranging from a few nanometers to an undefined level of micrometers. ENPs are resolved by two perpendicular flows—channel flow and cross-flow—in a thin, flat channel with a semi-permissive bottom wall membrane. The AF4 separation method offers several advantages over other isolation methods for ENP analysis, including being label-free, gentle, rapid (<1 h) and highly reproducible, as well as providing efficient recovery of analytes. Most importantly, in contrast to other available techniques, AF4 can separate ENPs at high resolution (1 nm) and provide a large dynamic range of size-based separation. In conjunction with real-time monitors, such as UV absorbance and dynamic light scattering (DLS), and an array of post-separation characterizations, AF4 facilitates the successful separation of distinct subsets of exosomes and the identification of exomeres. Although the whole procedure of cell culture and ENP isolation from the conditioned medium by ultracentrifugation (UC) can take ~3 d, the AF4 fractionation step takes only 1 h. Users of this technology will require expertise in the working principle of AF4 to operate and customize protocol applications. AF4 can contribute to the development of high-quality, exosome- and exomere-based molecular diagnostics and therapeutics.Zhang and Lyden describe a protocol for asymmetric-flow field-flow fractionation (AF4) to separate and characterize extracellular nanoparticles for investigation of their biogenesis, function and potential in molecular diagnostics and therapeutics.

The analysis of aggregates of therapeutic proteins is crucial in order to ensure efficacy and patient safety. Typically, the analysis is performed in the finished formulation to ensure that aggregates are not present. An important question is, however, what happens to therapeutic proteins, with regard to oligomerization and aggregation, after they have been administrated (i.e., in the blood). In this paper, the separation of whole blood, plasma, and serum is shown using asymmetric flow field-flow fractionation (AF4) with a minimum of sample pre-treatment. Furthermore, the analysis and size characterization of a fluorescent antibody in blood plasma using AF4 are demonstrated. The results show the suitability and strength of AF4 for blood analysis and open new important routes for the analysis and characterization of therapeutic proteins in the blood.

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.

Under laminar flow conditions, when no external forces are applied, particles are generally thought to follow fluid streamlines. Contrary to this perspective, we observe that flowing particles migrate across streamlines in a continuous, predictable, and accurate manner in microchannels experiencing laminar flows. The migration is attributed to lift forces on particles that are observed when inertial aspects of the flow become significant. We identified symmetric and asymmetric channel geometries that provide additional inertial forces that bias particular equilibrium positions to create continuous streams of ordered particles precisely positioned in three spatial dimensions. We were able to order particles laterally, within the transverse plane of the channel, with >80-nm accuracy, and longitudinally, in regular chains along the direction of flow. A fourth dimension of rotational alignment was observed for discoidal red blood cells. Unexpectedly, ordering appears to be independent of particle buoyant direction, suggesting only minor centrifugal contributions. Theoretical analysis indicates the physical principles are operational over a range of channel and particle length scales. The ability to differentially order particles of different sizes, continuously, at high rates, and without external forces in microchannels is expected to have a broad range of applications in continuous bioparticle separation, high-throughput cytometry, and large-scale filtration systems.

Exosomes participate in cancer metastasis, but studying them presents unique challenges as a result of their small size and purification difficulties. Asymmetrical field flow fractionation with in-line ultraviolet absorbance, dynamic light scattering, and multi-angle light scattering was applied to the size separation and characterization of non-labeled B16-F10 exosomes from an aggressive mouse melanoma cell culture line. Fractions were collected and further analyzed using batch mode dynamic light scattering, transmission electron microscopy and compared with known size standards. Fractogram peak positions and computed radii show good agreement between samples and across fractions. Ultraviolet absorbance fractograms in combination with transmission electron micrographs were able to resolve subtle heterogeneity of vesicle retention times between separate batches of B16-F10 exosomes collected several weeks apart. Further, asymmetrical field flow fractionation also effectively separated B16-F10 exosomes into vesicle subpopulations by size. Overall, the flow field flow fractionation instrument combined with multiple detectors was able to rapidly characterize and separate exosomes to a degree not previously demonstrated. These approaches have the potential to facilitate a greater understanding of exosome function by subtype, as well as ultimately allow for “label-free” isolation of large scale clinical exosomes for the purpose of developing future exosome-based diagnostics and therapeutics. Figure Flow path of exosome sample through the asymmetrical field flow fractionation instrument, detectors, and transmission electron microscope.

In asymmetrical flow field-flow fractionation (AF4), similar to other separation techniques, mass recovery and overloading require special attention in order to obtain quantitative results. We conducted a systematic study with five globular proteins of different molecular weight (36.7–669 kDa) and isoelectric point (4.0–6.5), and ultrafiltration membranes that are commonly used in aqueous AF4, regenerated cellulose (RC) and polyethersulfone (PES). Phosphate-buffered saline (PBS) with ionic strength 0.15 M and pH 7.2 was used as the carrier liquid in this study. The actual molecular weight cutoff (MWCO) was found to be higher than the nominal value and varied between membranes of different chemistry but the same nominal MWCO. Adsorption on the membrane was found to be dependent on the membrane chemistry (RC had lower adsorption compared to PES), and independent of the protein standard for the examined proteins. On the other hand, the mass overloading effects (i.e., higher retention times, peak broadening, and fronting peaks) were significantly more pronounced for γ-globulin than for the other proteins. The overloading effects could be rationalized with the increase of the local viscosity close to the membrane, depending on the properties of the proteins, and we derived theoretical equations that related the dependency of the migration velocity on the protein concentration through this non-ideal viscosity effect.

Biological small‐angle X‐ray scattering (SAXS) is a versatile and powerful technique for investigating the structural and biophysical properties of biologically and pharmaceutically relevant macromolecules and nanoparticles. SAXS offers detailed insights into macromolecular composition, size, shape and internal structure, while addressing key aspects such as oligomeric state, stability, molecular interactions, and conformational flexibility. Recently, asymmetrical‐flow field‐flow fractionation (AF4) was successfully coupled to SAXS, enabling online size‐based fractionation and analysis of polydisperse samples. This approach allows precise, size‐dependent characterization, offering significant advancements in the study of polydisperse systems. We have integrated an AF4 device at the P12 beamline at the European Molecular Biology Laboratory and implemented technical adaptations allowing full automation to make the system suitable for routine user access. We provide streamlined workflows and troubleshooting resources for both novice and advanced SAXS users thereby equipping them with clear guidance on performing AF4–SAXS measurements. The general principles of our set‐up are easily adaptable to other beamlines which have integrated (or are planning to integrate) a similar system. By coupling asymmetrical‐flow field‐flow fractionation to small‐angle X‐ray scattering (AF4–SAXS), we enable precise, size‐resolved analysis of polydisperse samples. Our automated AF4–SAXS system at the EMBL P12 beamline streamlines workflows, making advanced characterization accessible to both novice and experienced users, with principles adaptable to other facilities.

We report on a microfluidic particle-separation device that makes use of the asymmetric bifurcation of laminar flow around obstacles. A particle chooses its path deterministically on the basis of its size. All particles of a given size follow equivalent migration paths, leading to high resolution. The microspheres of 0.8, 0.9, and 1.0 micrometers that were used to characterize the device were sorted in 40 seconds with a resolution of ~10 nanometers, which was better than the time and resolution of conventional flow techniques. Bacterial artificial chromosomes could be separated in 10 minutes with a resolution of ~12%.

A method of analysis of silver nanoparticles (AgNPs) in chicken meat was developed. The homogenized chicken meat sample, which was spiked with AgNPs, was subjected to enzymolysis by Proteinase K for 40 min at 37 °C. Transmission electron microscopy and inductively coupled plasma mass spectrometry (ICP-MS) in single particle mode were used to characterize the number-based size distribution of AgNPs in the meat digestate. Because similar size distributions were found in the meat digestate and in the aqueous suspension of AgNPs used for spiking the meat, it was shown that no detectable dissolution of the AgNPs took place during the sample preparation stage. The digestate was injected into the asymmetric flow field flow fractionation (AF 4 ) -ICP-MS system, which enabled fractionation of nanoparticles from the remaining meat matrix, and resulted in one large peak in the fractograms as well as two smaller peaks eluting close to the void volume. The recovery of silver contained in the large AgNP peak was around 80 %. Size determination of AgNPs in the meat matrix, based on external size calibration of the AF 4 channel, was hampered by non-ideal (early elution) behavior of the AgNPs. Single particle ICP-MS was applied for determination of the number-based particle size distribution of AgNPs in collected fractions. The presented work describes for the first time the coupling of AF 4 and ICP-MS for AgNP separation in a food matrix.

While the exact health risks associated with nanoplastics are currently the focus of intense research, there is no doubt that humans are exposed to nanoplastics and that food could be a major source of exposure. Nanoplastics are released from plastic materials and articles used during food production, processing, storage, preparation, and serving. They are also likely to enter the food chain via contaminated water, air, and soil. However, very limited exposure data for risk assessment exists so far due to the lack of suitable analytical methods. Nanoplastic detection in food poses a great analytical challenge due to the complexity of plastics and food matrices as well as the small size and expectedly low concentration of the plastic particles. Multidetector field flow fractionation has emerged as a valuable analytical technique for nanoparticle separation over the last decades, and the first studies using the technique for analyzing nanoplastics in complex matrices are emerging. In combination with online detectors and offline analysis, multidetector field flow fractionation is a powerful platform for advanced characterization of nanoplastics in food by reducing sample complexity, which otherwise hampers the full potential of most analytical techniques. The focus of this article is to present the current state of the art of multidetector field flow fractionation for nanoplastic analysis and to discuss future trends and needs aiming at the analysis of nanoplastics in food. Graphical Abstract