Cyclical Electrical Field Flow Fractionation Research Articles

Insight into the formation and biological effects of natural organic matter corona on silver nanoparticles in water environment using biased cyclical electrical field-flow fractionation

Natural organic matter (NOM) readily interacts with nanoparticles, leading to the formation of NOM corona structures on their surface. NOM corona formation is closely related to the surface coatings and bioavailability of nanoparticles. However, the mechanism underlying NOM corona formation on silver nanoparticles (AgNPs) remains largely unknown due to the lack of effective analytical methods for identifying the changes in the AgNP surface. Herein, the separation ability of biased cyclical electrical field-flow fractionation (BCyElFFF) for same-sized polyvinyl pyrrolidone-coated and poly(ethylene glycol)-coated silver nanoparticles (AgNPs) with different electrophoretic mobilities was evaluated under various electrical conditions. Then, the mechanism behind the NOM corona formation on these AgNP surfaces was elucidated based on the changes in the elution time and off-line characterization of the collected fractions during their elution time in a BCyElFFF run. Finally, the survival rates of E. coli exposed to polyvinyl pyrrolidone-coated and poly(ethylene glycol)-coated AgNPs with or without NOM collected during repeated BCyElFFF runs were observed to increase with increasing NOM concentration, clearly demonstrating the negative effect of NOM corona structures on the bioavailability of AgNPs. These findings highlight the powerful separation and isolation ability of BCyElFFF in studying the transformation and fate of nanoparticles in aqueous environments.

Characterization of Human Glioblastoma versus Normal Plasma-Derived Extracellular Vesicles Preisolated by Differential Centrifugation Using Cyclical Electrical Field-Flow Fractionation.

Although many properties for small extracellular vesicles (sEVs, formerly termed "exosomes") isolated at ∼100 000g are known, a wide range of values are reported for their electrophoretic mobility (EM) measurements. This paper reports for the first time the effect of dilution on the EM of U87 glioblastoma cell-derived and plasma-derived sEVs and medium size EVs (mEVs, commonly termed "oncosomes") preisolated by differential centrifugation. Furthermore, the effect of resalting on the EM of sEVs and mEVs was evaluated. The EM of U87 sEVs and U87 mEVs showed an increase as the salt concentration decreased to 0.005% of the initial salt concentration. However, for the plasma sEVs and plasma mEVs, the electrophoretic mobility increased as the salt concentration decreased to 0.01% of the initial salt concentration and then increased to its initial value when the salt concentration decreased to 0.005% of the initial salt concentration. For both U87 and plasma sEVs and mEVs, the EM remained almost constant when the concentration of the particles changed and the salt concentration was kept the same as its initial value. This indicates that the EM of EVs is only a function of the salt concentration of the buffer and is independent of the concentration of the particles. The sEVs and mEVs were separated with cyclical ElFFF for the first time. The results indicate that ElFFF was able to fractionate the EVs, and a crescent-shaped trend was found for the retention time when the applied AC voltage was altered (increased).

Characterization and differential retention of Q beta bacteriophage virus-like particles using cyclical electrical field-flow fractionation and asymmetrical flow field-flow fractionation.

Virus-like particles (VLPs) are widely used in medicine, but can be difficult to characterize and isolate from aggregates. In this research, primarily cyclical electrical field-flow fractionation (CyElFFF) coupled with multi-angle light scattering (MALS), and dynamic light scattering (DLS) detectors, was used for the first time to perform size and electrical characterization of three different types of Q beta bacteriophage virus-like particles (VLPs): a blank Q beta bacteriophage which is denoted as VLP and two conjugated ones with different peptides. The CyElFFF results were verified with transmission electron microscopy (TEM). Asymmetrical flow field-flow fractionation (AF4) coupled with MALS was also applied using conditions similar to those used in the CyElFFF experiments, and the results of the two techniques were compared to each other. Using these techniques, the size and electrophoretic characteristics of the fractionated VLPs in CyElFFF were obtained. The results indicate that CyElFFF can be used to obtain a clear distribution of electrophoretic mobilities for each type of VLP. Accordingly, CyElFFF was able to differentially retain and isolate VLPs with high surface electric charge/electrophoretic mobility from the ones with low electric charge/electrophoretic mobility. Regarding the size characterization, the size distribution of the eluted VLPs was obtained using both techniques. CyElFFF was able to identify subpopulations that did not appear in the AF4 results by generating a shoulder peak, whereas AF4 produced a single peak. Different size characteristics of the VLPs appearing in the shoulder peak and the main peak indicate that CyElFFF was able to isolate aggregated VLPs from the monomers partially. Graphical abstract.

Exosome Isolation: Cyclical Electrical Field Flow Fractionation in Low-Ionic-Strength Fluids.

The influence of buffer substitution and dilution effects on exosome size and electrophoretic mobility were shown for the first time. Cyclical electrical field flow fractionation (Cy-El-FFF) in various substituted fluids was applied to exosomes and other particles. Tested carrier fluids of deionized (DI) water, 1× phosphate buffered saline (PBS), 0.308 M trehalose, and 2% isopropyl alcohol (IPA) influenced Cy-El-FFF-mediated isolation of A375 melanoma exosomes. All fractograms revealed a crescent-shaped trend in retention times with increasing voltage with the maximum retention time at ∼1.3 V AC. A375 melanoma exosome recovery was approximately 70-80% after each buffer substitution, and recovery was independent of whether the sample was substituted into 1× PBS or DI water. Exosome dilution in deionized water produced a U-shaped dependence on electrophoretic mobility. The effect of dilution using 1× PBS buffer revealed a very gradual change in electrophoretic mobility of exosomes from ∼-1.6 to -0.1 μm cm/s V, as exosome concentration was decreased. This differed from the use of DI water, where a large change from ∼-5.5 to -0.1 μm cm/s V over the same dilution range was observed. Fractograms of separated A375 melanoma exosomes in two substituted low-ionic-strength buffers were compared with synthetic particle fractograms. Overall, the ability of Cy-El-FFF to separate exosomes based on their size and charge is a highly promising, label-free approach to initially catalogue and purify exosome subtypes for biobanking as well as to enable further exosome subtype interrogations.

Effect of Ionic and Nonionic Carriers in Electrical Field-Flow Fractionation.

A major limitation of electrical field-flow fractionation (ElFFF) is the polarization of the electrodes that occurs when using an ionic carrier liquid. As there is great interest in using ElFFF with biological materials and biological materials typically have high ionic strengths and high osmotic concentrations, we explore the effect of concentration for phosphate buffered saline (PBS), a typical ionic medium for biological samples, and for two nonionic materials common in bioparticle analysis: isopropanol (IPA) and sucrose. Their effect on retention and separations in ElFFF for increasing concentrations was observed. The results suggest that modifying the carrier solution with PBS, sucrose, and/or IPA would enable characterization and separation of biological samples in ElFFF. Specifically, changes of elution time and electrical parameters such as current, conductivity, and bulk channel resistance were observed as functions of carrier ionic and osmotic strength for the different carrier additives. PBS can be used in the micromolar range, equivalent to about 0.1% 1× PBS (150 μM). These concentrations are far from the isotonic condition of PBS (∼ 150 mM) that is normally used with biological samples. However, the nonionic additive carriers IPA and sucrose show quality retention even when added in high concentrations. The results show that IPA could be used in ratios up to 60% and that sucrose can be used in concentrations up to 0.3 M. Concentrations of 2% IPA (0.26 M) and 0.30 M sucrose are biologically isotonic conditions (275-299 mOsm/kg), and retention was readily obtained in these conditions using both DC ElFFF and cyclical ElFFF (CyE1FFF). Carriers of this type may make it possible to use ElFFF with biological samples.

Biophysical Characterization of VLPs

Genetic Engineering & Biotechnology NewsVol. 35, No. 2 Bioprocessing TutorialBiophysical Characterization of VLPsAdvantages of Utilizing a Light-Scattering Toolbox in Vaccine DevelopmentDaniel SomeDaniel SomeDaniel Some, Ph.D., (E-mail Address: dsome@wyatt.com) is principal scientist at Wyatt Technology. Web: www.wyatt.comSearch for more papers by this authorPublished Online:13 Jan 2015https://doi.org/10.1089/gen.35.02.14AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byCurrent status and future challenges in transitioning to continuous bioprocessing of virus‐like particles30 June 2021 | Journal of Chemical Technology & Biotechnology, Vol. 97, No. 9Characterization and differential retention of Q beta bacteriophage virus-like particles using cyclical electrical field–flow fractionation and asymmetrical flow field–flow fractionation14 January 2020 | Analytical and Bioanalytical Chemistry, Vol. 412, No. 7Production of Well-Characterized Virus-like Particles in an Escherichia coli-Based Expression Platform for Preclinical Vaccine Assessments14 April 2016 Volume 35Issue 2Jan 2015 Information© 2015 by GEN PublishingTo cite this article:Daniel Some.Biophysical Characterization of VLPs.Genetic Engineering & Biotechnology News.Jan 2015.28-28.http://doi.org/10.1089/gen.35.02.14Published in Volume: 35 Issue 2: January 13, 2015PDF download

Circuit modification in electrical field flow fractionation systems generating higher resolution separation of nanoparticles

Compared to other sub-techniques of field flow fractionation (FFF), cyclical electrical field flow fractionation (CyElFFF) is a relatively new method with many opportunities remaining for improvement. One of the most important limitations of this method is the separation of particles smaller than 100nm. For such small particles, the diffusion rate becomes very high, resulting in severe reductions in the CyElFFF separation efficiency. To address this limitation, we modified the electrical circuitry of the ElFFF system. In all earlier ElFFF reports, electrical power sources have been directly connected to the ElFFF channel electrodes, and no alteration has been made in the electrical circuitry of the system. In this work, by using discrete electrical components, such as resistors and diodes, we improved the effective electric field in the system to allow high resolution separations. By modifying the electrical circuitry of the ElFFF system, high resolution separations of 15 and 40nm gold nanoparticles were achieved. The effects of applying different frequencies, amplitudes and voltage shapes have been investigated and analyzed through experiments.

Biased Cyclical Electrical Field Flow Fractionation for Separation of Sub 50 nm Particles

Cyclical electrical field flow fractionation (CyElFFF) is a technique for characterizing and separating nanoparticles based on their size and charge using cyclical electric fields. The high diffusion rate of nanoparticles has prevented CyElFFF from being applicable to particles smaller than 100 nm. In this work, the diffusion challenges associated with nanoparticles was resolved using biased cyclical electric fields. This new method, biased cyclical electrical field flow fractionation (BCyElFFF), achieves baseline separation of 15 and 40 nm gold nanoparticles. Theoretical considerations show that the optimal resolution is achieved when the applied bias yields electrical transport that counteracts the diffusive transport of nanoparticles. BCyElFFF greatly extends separation capabilities of the cyclical electrical field flow fractionation to sub 50 nm nanoparticles and provides a powerful alternative to other separation and characterization techniques capable of separating nanoparticles smaller than 50 nm.

Separation of Magnetic Nanoparticles by Cyclical Electrical Field Flow Fractionation

In this study, the potential of Cyclical Electrical Field Flow Fractionation (CyEFFF) for the separation of magnetic nanoparticles is investigated. We demonstrated for the first time that by the application of appropriate voltage waveforms, one can separate gold nanoparticles with sizes less than 50 nm. By using suitable voltage waveforms, the detrimental effect of the particle diffusion is suppressed and particles in the range of 10 nms can be fractionated. In addition, it is shown that CyEFFF is capable of separating lipid and polystyrene sulfonate coated magnetite nanoparticles with the same hydrodynamic radius of 50 nm.

Characterization of Polymerized Liposomes Using a Combination of dc and Cyclical Electrical Field-Flow Fractionation

Characterization of polymerized liposomes (PolyPIPosomes) was carried out using a combination of normal dc electrical field-flow fractionation and cyclical electrical field-flow fractionation (CyElFFF) as an analytical technique. The constant nature of the carrier fluid and channel configuration for this technique eliminates many variables associated with multidimensional analysis. CyElFFF uses an oscillating field to induce separation and is performed in the same channel as standard dc electrical field-flow fractionation separation. Theory and experimental methods to characterize nanoparticles in terms of their sizes and electrophoretic mobilities are discussed in this paper. Polystyrene nanoparticles are used for system calibration and characterization of the separation performance, whereas polymerized liposomes are used to demonstrate the applicability of the system to biomedical samples. This paper is also the first to report separation and a higher effective field when CyElFFF is operated at very low applied voltages. The technique is shown to have the ability to quantify both particle size and electrophoretic mobility distributions for colloidal polystyrene nanoparticles and PolyPIPosomes.

A novel method for effective field measurements in electrical field‐flow fractionation

The electric field that drives separation and retention in electrical field flow fractionation (ElFFF) and cyclical electrical field-flow fractionation (CyElFFF) is a complex function of many parameters such as carrier ionic strength and pH, voltage, channel dimensions, flowrate, and electrode material. Currently there is no accurate or in situ method to measure the field during system operation. This paper introduces a technique to measure the effective electric field during ElFFF and CyElFFF operation using transient electrical spikes. With this technique we can determine the relationship between changes in carrier conductivity and flowrate during a run and their combined effect on effective field and retention in ElFFF. This technique can also be used to measure the voltage drop due to double layer capacitance in CyElFFF and the variation in effective field with frequency of the applied field. The measured effective fields for the CyElFFF and DC ElFFF techniques are also tested with a high ionic-strength buffer solution as carrier. For a high ionic-strength buffer, DC ElFFF generates a near-zero effective field (0.2% in 100 s), whereas CyElFFF can sustain much higher effective fields (~8%) even at relatively high voltages. The ability to measure the effective field allows for experiments to provide better data and for tuning and optimization of the separation run.

Nanoparticle Characterization by Cyclical Electrical Field-Flow Fractionation

In this work, the analytical potential of cyclical electrical field flow fractionation (CyElFFF) for nanomaterial and colloidal particle characterization has been experimentally demonstrated. Different operating parameters were investigated in order to evaluate their effect on the mechanisms of retention and fractionation power of CyElFFF. The voltage and frequency of the oscillating electrical field appeared to be the most influential parameters controlling the separation mode. Mobile phase flow rate was also found to be a key parameter controlling the fractionation efficiency. This work allowed the definition of operating conditions such that a reliable CyElFFF analysis could be performed on different nanoparticles on the basis of the direct comparison of their theoretical and experimental behavior. The results show that this technique in optimized conditions is a powerful tool for electrophoretic mobility based separation and characterization of various nanoparticles.

Optimization of cyclical electrical field flow fractionation

Cyclical electrical field flow fractionation (CyElFFF) is a variation on electrical field flow fractionation (ElFFF) where cyclical electrical fields are used instead of steady DC fields to increase the effective field experienced by particles in the flow channel. Even though the effective field increases more than 20-fold compared to normal ElFFF, the retention and resolution in CyElFFF has not been shown to be better than in ElFFF. In this paper we report how one can optimize operational parameters in CyElFFF to obtain good retention and resolution in CyElFFF. The effects of offset voltage, frequency, flowrate, concentration of particles and sample size on retention, resolution and retained peak/void peak ratio have been observed. The results obtained from these experiments were analyzed and suggestions have been made to improve both retention and resolution. A 4-fold improvement in retention without a significant increase in band broadening is reported.

Modeling of micro/nano particle separation in microchannels with field-flow fractionation

The cyclical electrical field-flow fractionation (CyElFFF) is a very promising separation technique for particles and biological molecules such as proteins, nucleic acids, viruses, bacteria, yeast cells, mammalian cells. But a clear understanding of the mechanism and performance prediction of this system under different operating parameters is far from completed. This research focuses on a computational investigation of particle behavior in a CyElFFF system by taking into account both electrokinetic effects and particle dynamics. The model was validated with both theory and experimental results. The effects of key parameters such as applied electric field strength and frequency, solution fluid flow rate, particle size, particle shape on separation process are addressed in a systematic way. The developed model can also be utilized in studying the behavior of spherical or non-spherical particles (such as nanowire, nanorod, and nanofiber) in other microfluidic systems.

Improved theory of cyclical electrical field flow fractionation

Previously reported theories for cyclical electrical field flow fractionation (CyElFFF) are severely limited in that they do not account for diffusion, steric, or electric double layer effects. Experiments have shown that these theories overpredict the retention of particles in CyElFFF. In this work, we present a model for prediction of steric, diffusion, and electrical effects. The electrical double layer effects are treated using a lumped electrical circuit model that accounts for the field shielding by the electrical double layer formed at the electrode-carrier interface. The electrical effects are shown to dominate retention times and outweigh the contributions of diffusion and particle size. Detailed results from the simulations are presented in this work, and a comparison between the theoretical and experimental results obtained from the retentions of polystyrene particle standards is presented in this paper. The models are shown to correctly predict the retention of the polystyrene standards in CyElFFF with a reasonable error, while existing models are shown to have significant failings.

Effect of Carrier Ionic Strength in Microscale Cyclical Electrical Field-Flow Fractionation

Recent work with cyclical electrical field-flow fractionation systems has shown promise for the technique as a separation and analysis tool, but little is understood about how the carrier composition in the system affects its capabilities. The electrical properties of microscale CyElFFF systems change when the carrier ionic conditions are altered, and it is well known that the effects of increasing ionic strength carriers on retention in normal ElFFF systems are severe. Specifically, retention levels fall significantly. Accordingly, this work seeks to understand the effect that increasing carrier ionic strength in CyElFFF has on nanoparticle retention in the channels. The retention of polystyrene particles in the CyElFFF microsystem is reported at various ionic strengths of ammonium carbonate and at a variety of pH levels. The experiments are compared to the theory of CyElFFF available in the literature. The results indicate that the ionic strength of the carrier has a significant impact on retention and that high ionic strength carrier solutions lead to poor performance of the CyElFFF system. These results have significant impact on the possible uses of the technique and its applications, especially in the biomedical arena.

Characterization of a microscale cyclical electrical field flow fractionation system

A microscale cyclical electrical field flow fractionation (CyElFFF) channel is characterized with regard to the effect of various operating parameters and comparison made to recent theoretical developments. Challenges associated with various operating conditions are reported along with some of the optimized operating parameters. The effect of retention wall choice, an offset voltage, relaxation steps, and flow rates, along with the basic operating parameters of voltage, frequency, and electrophoretic mobility are reported. Retention of polystyrene nanoparticle standards is accomplished and the first separations using this technique in a microscale system are also demonstrated. Relaxation steps and offset voltages are found to be effective in eliminating early peaks and in improving plate heights. Plate heights were also found to decrease with increasing flow rates, which is the opposite of the behavior seen in most existing chromatographic systems. The experimental results are compared to the analytical and empirical models of CyElFFF and found to be compatible. Suggestions are made for improving the separation and analysis methods used with CyElFFF.

Cyclical electrical field flow fractionation

Cyclical electrical field flow fractionation (Cy/ElFFF) is demonstrated in a standard electrical field flow fractionation (ElFFF) channel for the first time. Motivation for the use of alternating current (AC) fields in a traditionally direct current (DC) technique are discussed. The function of the system over a wide range of operating conditions is explored and challenges associated with various operating conditions reported. Retention of polystyrene nanoparticle standards is accomplished and the effect of varying parameters of the applied field, such as voltage and frequency, are explored. The first separations using this technique are demonstrated. The experimental results are compared to analytical models previously reported in the literature. The general trend of the experimental results is similar to those predicted in theoretical models and possible reasons for discrepancies are elucidated. Suggestions are made for improving the separation and analysis method, and possible applications explored.