Search

Extracellular vesicles (EVs) are emerging as crucial biomarkers in cancer diagnostics and therapeutics with their heterogeneity presenting both challenges and opportunities in prostate cancer research. However, existing methods for isolating and characterizing EV subtypes have been limited by inefficient separation and inadequate proteomic analysis. Here we show an optimized centrifugal microfluidic device, Exodisc, that efficiently isolates large quantities of EV subtypes from particle-enriched medium, enabling comprehensive proteomic analysis of small (EV-S, 20-200 nm) and large (EV-L, >200 nm) EVs. Using this device, we successfully separated EV-S and EV-L from prostate cancer cell lines LNCaP and PC3. Mass spectrometry-based proteomics revealed that EV proteins reflect parental cell characteristics more than EV size, with EV-L demonstrating increased expression of PSMA-correlated proteins. Our optimized protocol addresses challenges in EV isolation and characterization, providing a more effective method for studying cellular and molecular mechanisms of specific EV subtypes. This study extends the potential use of EVs as a liquid biopsy for cancer theranostics, paving the way for more precise isolation of EV subtypes and potentially leading to improved biomarker discovery and the development of personalized treatments.

Hydrodynamic chromatography (HDC) is a size-based liquid chromatographic technique with similar applications as size exclusion chromatography (SEC) and flow field flow fractionation (flow FFF). Separation in HDC is induced within the parabolic flow velocity profile in an open tube or in the interstitial spaces of a packed column, whereby analytes preferentially sample the various streamlines of flow based on size and as a result the larger analytes elute prior to the smaller ones. HDC experimental set-ups can utilize a range of detection methods, e.g., refractive index (RI), UV, multi-angle static light scattering (MALS), quasi-elastic light scattering (QELS), differential viscometry and inductively coupled plasma mass spectrometry (ICP-MS), coupled in various combinations. Here, we discuss the historical use of single-detector HDC for the determination of polymer/particle size using calibration standards, followed by the more modern-day approach of multi-detector HDC, which can be used for the determination of polymer/particle molar mass, size, shape and compactness. Finally, we provide a perspective on the potential and limitations of HDC as a technique for the characterization of polymers and particles. [ABSTRACT FROM AUTHOR]

Organelle size varies with normal and abnormal cell function. Thus, size-based particle separation techniques are key to assessing the properties of organelle subpopulations differing in size. Recently, insulator-based dielectrophoresis (iDEP) has gained significant interest as a technique to manipulate sub-micrometer-sized particles enabling the assessment of organelle subpopulations. Based on iDEP, we recently reported a ratchet device that successfully demonstrated size-based particle fractionation in combination with continuous flow sample injection. Here, we used a numerical model to optimize the performance with flow rates a factor of three higher than previously and increased the channel volume to improve throughput. We evaluated the amplitude and duration of applied low-frequency DC-biased AC potentials improving separation efficiency. A separation efficiency of nearly 0.99 was achieved with the optimization of key parameters-improved from 0.80 in previous studies (Ortiz et al. Electrophoresis, 2022;43;1283-1296)-demonstrating that fine-tuning the periodical driving forces initiating the ratchet migration under continuous flow conditions can significantly improve the fractionation of organelles of different sizes., (© 2023 Wiley-VCH GmbH.)

Abstract In the characterization of nanomaterials, size and size distribution are among the most important parameters. A high-resolution technique is essential for the accurate determination of size distribution for a polydisperse nanoparticle suspension. Although there are many size-based separation techniques available for this purpose, the open channel designs of capillary electrophoresis (CE) and asymmetric flow field-flow fractionation (AF4) provide excellent resolution and sample recovery. AF4 is a diffusion-based separation technique for colloidal particles with an analytical range from a few nanometers to several microns. Capillary electrophoresis separates particles based on their electrophoretic mobility (charge to size ratio) making CE applicable for charged particles. These two techniques can be used for the size-based separation of metallic nanoparticles depending on the surface charge and physicochemical properties of the surrounding medium. The purpose of this work is to provide a comprehensive discussion and comparison of CE and AF4 for the determination of the size and size distribution of engineered metallic nanoparticles. Highlights • The separation of nanoparticles using capillary electrophoresis and asymmetric flow field-flow fractionation are presented. • Recent advances and development of CE and AF4 are reviewed. • Limitations of each method for analysis of engineered metallic nanoparticles are elaborated. • The advantages and disadvantages of CE and AF4 are discussed in detail. [ABSTRACT FROM AUTHOR]

Harmful algal blooms (HABs) are a recurring phenomenon along all continents, posing a global threat, particularly due to shellfish poisoning and public health, and their timely monitoring is vital for safeguarding the blue economy. The ability to efficiently isolate species of interest, such as harmful dinoflagellates, is a key process for HABs monitoring which is particularly complex because of the typically high diversity of marine microalgae communities. This study introduces a simple spiral microchannel device using inertial microfluidics to separate and concentrate microalgae based on size. This device effectively concentrates microalgae, achieving up to 5.8-fold fluorescence increase after 3 cycles in the spiral and isolates target species with <6 % cell loss. A continuous enrichment method demonstrated a 2.85-fold increase in fluorescence signal for Alexandrium minutum after 80 min. This method proved suitable to enhance the sensitivity of devices designed to detect harmful phytoplankton species' early blooms. [Display omitted] • Technology with high impact in the earlier screen of harmful microalgae blooms effect • Microalgae cells enrichment method up to 5.8-fold increase • Cell separation size modulated by tuning the flow velocity with cell loss bellow to 6 % • First demonstration of the detection of very few cells from microalgae toxic species per seawater litter • Long-term monitoring the high diversity of phytoplankton communities [ABSTRACT FROM AUTHOR]

• Numerical investigation of ternary particle separation using DEP was presented. • A new code was developed in OpenFOAM to simulate particles movement. • The developed code was validated by performing experimental tests. • Various parameters were investigated to achieve the proper design. • Particles were separated with excellent separation efficiency and purity (100%) In recent years, microfluidic-based particle/cell manipulation techniques have catalyzed significant advances in several fields of science. As an efficient, precise, and label-free particle/cell manipulation technique, dielectrophoresis (DEP) has recently attracted widespread attention. This paper presents the design and investigation of a straight sheathless 3D microchannel with a wall-mounted trapezoidal obstacle for continuous-flow separation of three different populations of polystyrene (PS) particles (5, 10 and 20 µm) using DEP. An OpenFOAM code is developed to simulate and investigate the movement of particles in the microchannel. Then, the code is validated by performing various experimental tests using a microdevice previously fabricated in our lab. By comparing the numerical simulation results with the experimental tests, it can be claimed that the newly developed solver is highly accurate, and its results agree well with experimental tests. Next, the effect of various operational and geometrical parameters such as obstacle height, applied voltage, electrode pairs angle, and flow rate on the efficient focusing and separation of particles are numerically investigated. The results showed that efficient particle separation could only be achieved for obstacle heights of more than 350 µm. Furthermore, the appropriate voltage range for efficient particle separation is increased by decreasing the electrode angle as well as increasing the flow rate. Moreover, the results showed that by employing the appropriate channel design and operational conditions, at a maximum applied voltage of 10V, a sample flow rate of 2.5 μ L / min could be processed. The proposed design can be beneficial for integrating with lab-on-a-chip and clinical diagnosis applications due to advantages, such as simple design, no need for sheath flow, the simultaneous ternary separation of particles, and providing precise particle separation. [ABSTRACT FROM AUTHOR]

Viruses protect their genomes by enclosing them into protein capsids that sometimes contain lipid bilayers that either reside above or below the protein layer. Controlled dissociation of virions provides important information on virion composition, interactions, and stoichiometry of virion components, as well as their possible role in virus life cycles. Dissociation of viruses can be achieved by using various chemicals, enzymatic treatments, and incubation conditions. Asymmetrical flow field-flow fractionation (AF4) is a gentle method where the separation is based on size. Here, we applied AF4 for controlled dissociation of enveloped bacteriophage φ6. Our results indicate that AF4 can be used to assay the efficiency of the dissociation process and to purify functional subviral particles.

Reprocessing of high-level waste is a key step in advancing sustainable energy systems. The development of efficient chelating agents for trivalent f-block metal ions is essential for increasing the efficiency of nuclear-waste remediation and extractive hydrometallurgy of rare-earth elements. Although computer-aided screening could lead to a more rapid discovery of superior ligands, an accurate theoretical description of the solvation effects for trivalent metal ions is currently a stumbling block in qualitative predictions for selectivity trends along the lanthanide series. In this work, we propose a robust model to describe the differential effect of solvation in the competitive binding of a ligand with lanthanides by including weakly coordinated counterions in the complexes of more than a +1 charge. The success of this approach in quantitatively reproducing aqueous selectivities demonstrates its potential for the design and screening of new ligands for efficient size-based separation. [ABSTRACT FROM AUTHOR]

In this paper, we utilize the light-induced dielectrophoretic technique to realize the accurate control of nanowires, including positioning of single nanowires, separation of different-sized nanowires, and spacing regulation of uniform nanowire-arrays. Nanowires can exactly stabilize at the center of a regular geometric light-spot with a slight eccentricity of 2.1 ± 0.64%. Additionally, nanowires of different lengths (∼6 μm and ∼12 μm) are separated individually by a light-bar with a proper traveling velocity, which is measured and selected according to an acceleration-displacement measurement method. Based on this, uniform nanowire-arrays are assembled instantly, and emerge an equidistant distribution. In reality, the array spacing-distance presents a frequency dependence, and can be automatically adjusted from 6 to 21 μm for two-wire array and 7–16 μm for three-wire array, respectively. The accurate positioning and orderly management of nanowires may potentially increase the efficiency of nanowire assembly, and be contributed to the nano-device building. [ABSTRACT FROM AUTHOR]

Carbon nanotubes (CNTs) are prominent candidates for the separation of various polar and non-polar liquid mixtures. Numerous studies report the application of CNTs for isolating the binary aqueous liquid mixtures, but very few demonstrate the separation of organic liquid mixtures. Here, we investigate the selective permeation of cosolvents such as ethanol, isopropanol, n -butanol, acetone, and tetrahydrofuran into the CNTs from the binary mixtures of benzene using molecular dynamics simulations. Our findings reveal that extremely small-sized (11,0) CNTs achieve almost 100% cosolvent capture selectivity in all five equimolar binary mixtures. In considerably bigger-sized CNTs such as (20,0), benzene accumulates inside along with the cosolvent, however, forms a well-distinguishable layer close to the interior wall of CNTs analogous to the second layer of a double-walled CNT. The results confirm that the liquid capture and selectivity are governed by the polarity and molecular size of the solvents as well as the CNT diameters. [ABSTRACT FROM AUTHOR]

Quantum dot (QD) emits light of characteristic wavelength which depends on the size of the QD particles, and thus the quality control of QD requires accurate determination of its size distribution. Field-flow fractionation (FFF) is a separation technique that is well suited for the characterization of colloidal particles. In this study, CdS-QD particles were synthesized by a simple one-step γ-ray irradiation. Then asymmetrical flow FFF (AsFlFFF) and sedimentation FFF (SdFFF) were tested as tools for characterization of the particles. The single crystallite size of CdS-QD was measured to be about 4 nm by X-ray diffraction (XRD), UV-VIS absorption spectroscopy, and transmission electron microscopy (TEM). TEM images indicate that the particles were agglomerated to form larger secondary particles (∼ 110 nm). Effects of the various experimental parameters of FFF including the type of dispersing agent, the ionic strength of the carrier liquid, and strength of external field were investigated. It was found that, among the dispersing agents tested, a mixture of anionic and nonionic surfactants tends to give better elution of CdS particles than anionic or nonionic surfactants. Results suggest that FFF can provide some advantages over dynamic light scattering (DLS) or TEM for size characterization of CdS particles, and with a proper optimization, FFF (AsFlFFF or SdFFF) could become a useful tool for stability study as well as size analysis of nanoparticles such as the CdS particles synthesized in this study. [ABSTRACT FROM AUTHOR]

Heterogeneity in organelle size has been associated with devastating human maladies such as neurodegenerative diseases or cancer. Therefore, assessing the size-based subpopulation of organelles is imperative to understand the biomolecular foundations of these diseases. Here, we demonstrated a ratchet migration mechanism using insulator-based dielectrophoresis in conjunction with a continuous flow component that allows the size-based separation of submicrometer particles. The ratchet mechanism was realized in a microfluidic device exhibiting an array of insulating posts, tailoring electrokinetic and dielectrophoretic transport. A numerical model was developed to elucidate the particle migration and the size-based separation in various conditions. Experimentally, the size-based separation of a mixture of polystyrene beads (0.28 and 0.87 μ $\umu $ m) was accomplished demonstrating good agreement with the numerical model. Furthermore, the size-based separation of mitochondria was investigated using a mitochondria mixture isolated from HepG2 cells and HepG2 cells carrying the gene Mfn-1 knocked out, indicating distinct size-related migration behavior. With the presented continuous flow separation device, larger amounts of fractionated organelles can be collected in the future allowing access to the biomolecular signature of mitochondria subpopulations differing in size., (© 2022 Wiley-VCH GmbH.)

• An FI-AF4 separation method for dextran nanoparticles was developed. • FI-AF4 coupled to the DLS detector showed the whole distribution of nanoparticles. • The enhanced resolution of the FI-AF4 technique was proven. • Batch-mode DLS was not suitable and provided misleading information. • The good reproducibility of the particle preparation method was demonstrated. Accurate determinations of particle size and particle size distribution (PSD) are essential to achieve the clinical translation of medical nanoparticles (NPs). Herein, dextran-based NPs produced via a water-in-oil emulsification/crosslinking process and developed as nanomedicines were studied. NPs were first characterized using traditional batch-mode techniques as dynamic light scattering (DLS) and laser diffraction. In a second step, their analysis by frit-inlet asymmetrical flow field-flow fractionation (FI-AF4) was explored. The major parameters of the AF4 procedure, namely, crossflow, detector flow, crossflow decay programming and relaxation time were set up. The sizes of the particle fractions eluted under optimized conditions were measured using DLS as an online detector. We demonstrate that FI-AF4 is a powerful method to characterize dextran-NPs in the 200 nm -1 µm range. It provided a more realistic and comprehensive picture of PSD, revealing its heterogenous character and clearly showing the ratio of different populations in the sample, while batch-mode light scattering techniques only detected the biggest particle sizes. [ABSTRACT FROM AUTHOR]

• A separation for selectively collecting self-repairable particles in monodispersity. • Precipitated squid ring teeth inspired tandem repeat protein particles were tested. • The particles showed aqueous swelling capability that could enable delivery of drug. • Sorting by size and morphology was performed for the next-generation drug delivery. Recently, insoluble protein particles have been increasingly investigated for artificial drug delivery systems due to their favorable properties, including programmability for active drug targeting of diseases as well as their biocompatibility and biodegradability after administration. One of the biggest challenges is selectively collecting desirable self-repairable particles in the spherical morphology with monodispersity to enable consistent levels and rates of drug loading and release. Therefore, technology that allows sorting of protein particles with respect to size and morphology will enhance the design and production of next-generation drug delivery materials. Here, we introduce a dielectrophoretic (DEP) separation technique to selectively isolate spherical protein particles from a mixture of randomly shaped particles. We tested this approach by applying it to a mixture of precipitated squid ring teeth inspired tandem repeat protein particles with diverse sizes and morphologies. The DEP trapping system enabled us to isolate specific-sized, spherical protein particles out of this mixture: after separation, the fraction of 2 µm and 4 µm spherical particles was increased from 28.64% of mixture to 80.53% and 74.02% with polydispersity indexes (PDIs) decreased from 0.93 of mixture to 0.19 and 0.09, respectively. The protein particles show high aqueous swelling capability (up to 74% by mass) that could enable delivery of drug solutions. This work is intended to inspire the future development of biocompatible drug-delivery systems. [ABSTRACT FROM AUTHOR]

Asymmetrical flow field-flow fractionation (AF4) separates sample components based on their sizes in the absence of a stationary phase. It is well suited for high molecular weight samples such as virus-sized particles. The AF4 experiment can potentially separate molecules within a broad size range (~103−109 Da; particle diameter from 2 nm to 0.5−1 μm). When coupled to light scattering detectors, it enables rapid assays on the size, size distribution, degradation, and aggregation of the studied particle populations. Thus, it can be used to study the quality of purified viruses and virus-like particles. In addition to being an advanced analytical characterization technique, AF4 can be used in a semi-preparative mode. Here, we summarize and provide examples on the steps that need optimization for obtaining good separation with the focus on virus-sized particles. [ABSTRACT FROM AUTHOR]

Viruses protect their genomes by enclosing them into protein capsids that sometimes contain lipid bilayers that either reside above or below the protein layer. Controlled dissociation of virions provides important information on virion composition, interactions, and stoichiometry of virion components, as well as their possible role in virus life cycles. Dissociation of viruses can be achieved by using various chemicals, enzymatic treatments, and incubation conditions. Asymmetrical flow field-flow fractionation (AF4) is a gentle method where the separation is based on size. Here, we applied AF4 for controlled dissociation of enveloped bacteriophage φ6. Our results indicate that AF4 can be used to assay the efficiency of the dissociation process and to purify functional subviral particles. [ABSTRACT FROM AUTHOR]

Circulating tumor cells (CTCs) are tumor cells that originate from primary cancer tissues, enter the bloodstream in the body, and metastasize to the other organs. Simple and convenient methods for their detection, capture, and recovery from the blood of cancer patients would be highly desirable. We report here on a simple and convenient methodology to trap, culture, and re-collect cancer cells, the sizes of which are greater than those of normal hematologic cells, by the use of glass-bead filters (GBFs). We prepared GBFs with a diameter of 24 mm and thicknesses of 0.4 mm and 1.2 mm, with well-defined pores, by sintering round-shaped glass beads (diameter: 63-106 μm). A small integrated glass-bead filter (iGBF) with a diameter of ca. 9.6 mm for the use in filtering a small volume of blood was also designed and prepared. Using GBF and iGBF, it was possible to efficiently capture mouse Lewis lung carcinoma cells expressing green fluorescent protein spiked in saline/blood by single and repeated (circulation) filtrations in in vitro experiments with very small amounts of red blood cells being captured. In addition, we successfully captured B16 CTCs from the blood of a B16 melanoma metastasis mouse model by iGBF. Cancer cells/CTCs captured on/in the GBF could be cultured and efficiently recovered from the filters. Filtration by GBF had negligible effect on the adherent and proliferative characteristics of cancer cells. Simple and convenient methods for the capture, culture, and re-collection of CTCs by GBF along with flexibility of GBF, which permits them to be molded into suitable architectures having the desired shape and size, should be useful for early and convenient diagnosis and treatment of cancer and related diseases.

We report on a facile method to stabilize colloidal self-assembled (CSA) nanoparticles packed in microchannels for high speed size-based separation of proteins. Silica nanoparticles, self-assembled in a network of microfluidic channels, were stabilized with a methacrylate polymer prepared in situ through photopolymerization. The entrapment conditions were investigated to minimize the effect of the polymer matrix on the structure of the packing and the separation properties of the CSA beds. SEM shows that the methacrylate matrix links the nanoparticles at specific sphere-sphere contact points, improving the stability of the CSA structure at high electric fields (up to at least 1800 V/cm), allowing fast and efficient separation. The %RSD of the protein migration times varied between 0.3 and 0.5% (n = 4, in 1 day) and <0.83% over a period of 7 days (n = 28 runs) in a single device, at high field strength. The overall %RSD of protein migration times from chip-to-chip across a single fabrication run was 4.3% (n = 3) and between fabrication runs was 11% (n = 35), with 87% fabrication yield, demonstrating reproducible packing and entrapment behavior. The optimized entrapped CSA beds demonstrated better separation performance (plate height, H ∼ 200 nm) than similarly prepared on-chip CSA beds without the polymer entrapment. Polymer-entrapped CSA beds also exhibited superior protein resolving power: the minimum resolvable molecular weight difference of proteins in the polymer-entrapped CSA bed is 0.6 kDa versus ∼9 kDa for the native silica CSA bed (i.e. without polymer entrapment)., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Circulating tumor cells (CTCs) are promising markers to determine cancer patient prognosis and track disease response to therapy. We present a multi-stage microfluidic device we have developed that utilizes inertial and Dean drag forces for isolating CTCs from whole blood. We demonstrate a 94.2% ± 2.1% recovery of cancer cells with our device when screening whole blood spiked with MCF-7 GFP cells.