Free-flow Electrophoresis Research Articles

Size separation of graphene oxide using preparative free-flow electrophoresis

Graphene oxide nanosheets often bear a wide size distribution. However, it is critical to have nanosheets with narrow size distribution for their unique size-dependent physiochemical properties, and nanosheets with a narrow size distribution are the cornerstones for application. Therefore, efficient separation methods of graphene nanosheets have been given considerable attention in many scientific areas recently. Free-flow electrophoresis is extensively used in the separation and purification of biological molecules with continuous flow separation. The charged graphene oxide nanosheets to some extent are very close in size to biological molecules and share similarity in motion behavior in an electric field. Thus, in the present work, we present a new and simple means to separate graphene oxide nanosheets into more mono-dispersed size groups by using the free-flow electrophoresis technique. By optimizing the separation conditions, we were able to obtain graphene oxide sheets with narrow size distribution. The separated samples were characterized by atomic force microscopy, and the size measurements were made by using the software "Image Pro Plus." In addition, a brief discussion is also given into the theoretic background of the separation of graphene oxide according to the size by the technique of preparative free-flow electrophoresis.

Gas removal in free-flow electrophoresis using an integrated nanoporous membrane

The performance of continuous microfluidic free-flow electrophoresis (μFFE) is often compromised by the formation of gaseous products caused by electrolysis of water. We show that this adverse effect can be overcome by employing a nanoporous polytetrafluoroethylene (PTFE) membrane attached to a μFFE system which results in efficient removal of any gases formed. The respective assembly was manufactured via laser cutting and lamination. The complete microfluidic FFE chips consist of five layers, viz. (a) two supporting layers, one made of an adhesive transfer foil and the other from poly(ethylene terephthalate), (b) a hydrophobic membrane, (c) a microfluidic structure in a layer of PTFE, and (d) a bottom glass slide. Such a platform warrants a stable flow of electric current over hours of operation at electric field strength of around 500 V∙cm‾1. This is in contrast to conventional FFE microchips where the current decreases to zero within a few minutes (using the same separation parameters). Micropreparative separation of a mixture of three fluorophores was successfully accomplished continuously over 3 h using this micro-FFE chip and was not accompanied by any disturbances caused by formation of gases.

Correction: Corrigendum: Ion concentration polarization-based continuous separation device using electrical repulsion in the depletion region

We proposed a novel separation method, which is the first report using ion concentration polarization (ICP) to separate particles continuously. We analyzed the electrical forces that cause the repulsion of particles in the depletion region formed by ICP. Using the electrical repulsion, micro- and nano-sized particles were separated based on their electrophoretic mobilities. Because the separation of particles was performed using a strong electric field in the depletion region without the use of internal electrodes, it offers the advantages of simple, low-cost device fabrication and bubble-free operation compared with conventional continuous electrophoretic separation methods, such as miniaturizing free-flow electrophoresis (μ-FFE). This separation device is expected to be a useful tool for separating various biochemical samples, including cells, proteins, DNAs and even ions.

Experimental study on the optimization of general conditions for a free-flow electrophoresis device with a thermoelectric cooler.

With a given free-flow electrophoresis device, reasonable conditions (electric field strength, carrier buffer conductivity, and flow rate) are crucial for an optimized separation. However, there has been no experimental study on how to choose reasonable general conditions for a free-flow electrophoresis device with a thermoelectric cooler in view of Joule heat generation. Herein, comparative experiments were carried out to propose the selection procedure of general conditions in this study. The experimental results demonstrated that appropriate conditions were (i) <67 V/cm electric field strength; (ii) lower than 1.3 mS/cm carrier buffer conductivity (Tris-HCl: 20 mM Tris was titrated by HCl to pH 8.0); and (iii) higher than 3.6 mL/min carrier buffer flow rate. Furthermore, under inappropriate conditions (e.g. 400 V voltage and 40 mM Tris-HCl carrier buffer), the free-flow electrophoresis separation would be destroyed by bubbles caused by more Joule heating. Additionally, a series of applications under the appropriate conditions were performed with samples of model dyes, proteins (bovine serum albumin, myoglobin, and cytochrome c), and cells (Escherichia coli, Streptococcus thermophilus, and Saccharomyces cerevisiae). The separation results showed that under the appropriate conditions, separation efficiency was obviously better than that in the previous experiments with randomly or empirically selected conditions.

Separation of basic proteins from Leishmania using a combination of Free flow electrophoresis (FFE) and 2D electrophoresis (2-DE) under basic conditions.

Basic proteins, an important class of proteins in intracellular organisms such as Leishmania, are usually underrepresented on 2D gels. This chapter describes a method combining basic proteins fractionation using Free flow electrophoresis in isoelectric focusing mode (IEF-FFE) followed by protein separation using two-dimensional gel electrophoresis (2-DE) in basic conditions. The combination of these two techniques represents a great improvement for the visualization of Leishmania proteins with basic pI using 2D gels.

Rapid isoelectric point determination in a miniaturized preparative separation using jet-dispensed optical pH sensors and micro free-flow electrophoresis.

Herein, the fabrication, characterization, calibration, and application of integrated microfluidic platforms for fast isoelectric point (pI) determinations via free-flow electrophoresis with integrated inkjet-printed fluorescent pH sensor microstructures are presented. These devices allow one to determine the pI of a biomolecule from a sample mixture with moderately good precision and without addition of markers in typically less than 10 s total separation and analysis time. Polyhydroxyethyl methacrylate (pHEMA) hydrogels were covalently coupled with fluorescein and hydroxypyrene trisulfonic acid (HPTS)-based pH probes. These were piezoelectrically jet-dispensed onto acrylate-modified glass as pH sensor microarrays with a diameter of 300-600 μm and thicknesses of 0.4-2.4 μm with high spatial accuracy. Microchip fabrication and integration of these pH sensor arrays was realized by multistep liquid-phase photolithography from oligoethylene glycol precursors resulting in glass-based microfluidic free-flow isoelectric focusing (μFFIEF) chips with integrated pH observation capabilities. The microchips were characterized with regard to pH sensitivity, response times, photo-, and flow stability. Depending on the sensor matrix, they allowed IEF within a pH range of roughly 5.5-10.5 with good sensitivity and fast response times. These microchips were used for FFIEF of small molecule markers and several protein mixtures with simultaneous monitoring of local pH. This allowed the determination of their pI via multispectral imaging of protein and pH sensor fluorescence without addition of external markers. Obtained pI's were generally in good agreement with known data, demonstrating the applicability of the method for pI determination in micropreparative procedures within a time frame of a few seconds only.

Proteomic analysis of metacyclogenesis in Leishmania infantum wild-type and PTR1 null mutant

The parasite Leishmania exhibits a dimorphic life cycle with promastigotes found in the insect vector and amastigotes residing within mammalian macrophages. In the insect, promastigotes undergo metacyclogenesis with two main stages, the procyclic and metacyclic stages. Reduced pterins, provided by the pteridine reductase PTR1, are important factors controlling metacyclogenesis. We applied here the liquid-based free-flow electrophoresis technique for the analysis of L. infantum wild-type and PTR1 null-mutant procyclic and metacyclic proteomes. Significant modulations in energy metabolism, antioxidant and stress-related defences, genetic information processing, protein degradation and motility were observed between procyclic and metacyclic forms, in addition to numerous hypothetical proteins.

Reducing pH gradients in free-flow electrophoresis.

Small-volume continuous-flow synthesis (small-volume CFS) offers a number of benefits for use in small-scale chemical production and exploratory chemistry. Typically, small-volume CFS is followed by discontinuous purification; however, a fully continuous synthesis-purification combination is more attractive. Milli free-flow electrophoresis (mFFE) is a promising continuous-flow purification technique that is well suited for integration with small-volume CFS. The purification stability of mFFE, however, needs to be significantly improved before it can be feasible for this combination. One of the major sources of instability of mFFE is attributed to the ions produced as a result of electrolysis. These ions can form pH and conductivity gradients in mFFE, which are detrimental to separation quality. The severity of these gradients has not been thoroughly studied in mFFE. In this paper, we have experimentally demonstrated that detrimental pH gradients occur at flow rates of 8 mL/min and less, and electric field strengths of 25 V/cm and greater. To decrease the pH gradients, it is necessary to evacuate H(+) and OH(-) as soon as they are generated; this can be done by increasing local hydrodynamic flow rates. We calculated the necessary flow rate, to be applied at the electrode, which can effectively wash away both ions before they can cause a detrimental pH gradient. These optimized flow rates can be attained by designing a device that incorporates deep channels. We have confirmed the effectiveness of these channels using a prototyped device. The new design allows mFFE users to work over a wider range of flow-rate and electric-field conditions without experiencing significant changes in pH.

Negative‐pressure‐induced collector for a self‐balance free‐flow electrophoresis device

Uneven flow in free-flow electrophoresis (FFE) with a gravity-induced fraction collector caused by air bubbles in outlets and/or imbalance of the surface tension of collecting tubes would result in a poor separation. To solve these issues, this work describes a novel collector for FFE. The collector is composed of a self-balance unit, multisoft pipe flow controller, fraction collector, and vacuum pump. A negative pressure induced continuous air flow rapidly flowed through the self-balance unit, taking the background electrolyte and samples into the fraction collector. The developed collector has the following advantages: (i) supplying a stable and harmonious hydrodynamic environment in the separation chamber for FFE separation, (ii) effectively preventing background electrolyte and sample flow-back at the outlet of the chamber and improving the resolution, (iii) increasing the preparative scale of the separation, and (iv) simplifying the operation. In addition, the cost of the FFE device was reduced without using a multichannel peristaltic pump for sample collection. Finally, comparative FFE experiments on dyes, proteins, and cells were carried out. It is evident that the new developed collector could overcome the problems inherent in the previous gravity-induced self-balance collector.

Comprehensive multidimensional separations of peptides using nano-liquid chromatography coupled with micro free flow electrophoresis.

The throughput of existing liquid phase two-dimensional separations is generally limited by the peak capacity lost due to under sampling by the second dimension separation as peaks elute off the first dimension separation. In the current manuscript, a first dimension nanoliquid chromatography (nLC) separation is coupled directly with a second dimension micro free flow electrophoresis (μFFE) separation. Since μFFE performs continuous separations, no complicated injection or modulation is necessary to couple the two techniques. Analyte peaks are further separated in μFFE as they elute off the nLC column. A side-on interface was designed to minimize dead volume in the nLC × μFFE interface, eliminating this as a source of band broadening. A Chromeo P503 labeled tryptic digest of BSA was used as a complex mixture to assess peak capacity. 2D nLC × μFFE peak capacities as high as 2,352 could be obtained in a 10 min separation window when determined according to the product of the first and second dimension peak capacities. After considering the orthogonality of the two separation modes and the fraction of separation space occupied by peaks, the usable peak capacity generated was determined to be 776. The 105 peaks/min generated using 2D nLC × μFFE was nearly double the previously reported maximum peak capacity production rate achieved using online LC × LC.

Two Benches PDMS Free Flow Electrophoresis Chip

This paper puts forward a kind of two benches structure of the glass-PDMS free flow electrophoresis chip. We use die casting method to produce microstructure, and use PDMS as its structure layer. The mold is made in tape-PMMA by micro cementing technology. During the progress of PDMS curing, gold wires are directly integrated in the electrode groove to form the chips electrodes. PDMS and glass are sealed in natural bonding method. Chips glass surface of separation chamber realizes the modification through sticking polyimide tape. Visual technology is conducted for chip electrophoresis separation. The sample is the mixture of methyl green and Rhodamine B. The separation effect of methyl green and Rhodamine B is obvious when the electric field is 31 V/cm. the "two benches" between the electrode groove and the separation chamber block the electrolytic bubbles into the separation chamber. When the separation chamber height is 70 μm and the electric field is 50 V/cm, the air bubbles can be glidingly discharged from electrode groove. The samples steady separation time is more than 2 hours, and this free flow electrophoresis chip realizes continuous work.

A method-of-moments formulation for describing hydrodynamic dispersion of analyte streams in free-flow zone electrophoresis

In this work, a method-of-moments formulation has been presented for estimating the dispersion of analyte streams as they migrate through a free-flow zone electrophoresis (FFZE) channel under laminar flow conditions. The current analysis considers parallel-plate based FFZE systems with an applied pressure-gradient along the channel length for sample and carrier electrolyte transport, and an external electric field in the transverse direction for enabling the electrophoretic separation. A closed-form expression has been derived using this mathematical approach for describing the spatial variance of sample streams as a function of their position in the separation chamber at steady state. This expression predicts that the hydrodynamic dispersion component in an FFZE assay scales as Pex2 where Pex denotes the Péclet number based on the analyte's transverse electrophoretic migration velocity rather than its longitudinal pressure-driven flow speed as expected in transport processes induced by a pressure-gradient. Interestingly however, the coefficient multiplying this dimensionless group, i.e., 1/210, is identically equal to the constant preceding the square of the relevant Péclet number in the latter case (i.e., Péclet number based on the longitudinal flow speed). It must be noted that while the mathematical analysis reported in this work is only valid for FFZE systems in the absence of any unwanted Joule heating, pressure-driven cross-flow and/or differences in the electrical conductivity between the sample and carrier electrolyte, it can also be applied to numerically estimate the effect of these factors on the separation resolution of the assay.

Separation and detection of urinary proteins by two-dimensional electrophoresis on the microfluidic chip

A novel separating and detecting method of urinary proteins by non-gel sieving electrophoresis–free flow electrophoresis on the microfluidic chip was proposed in this article. Human serum albumin and human transferrin labeled by fluorescein isothiocyanate were mixed and taken as the synthesized urinary protein samples. Factors affected on separation, such as the rate of electric fields applied in two dimension, the addition in the buffer, the concentration, and pH of buffer, were optimized, that is, 10% (w/v) dextran, 0.5% ethanediamine, 0.2 mol L−1 Tris–boric acid, pH 9.3, the ratio of the applied electric field in two dimensions 1:4.8, respectively. Fluorescein isothiocyanate–human serum albumin and fluorescein isothiocyanate–transferrin were efficiently separated in 2 min, and the resolution of two proteins achieved was 2.2 by the designed microfluidic chip. Then, the urine samples of nephropathy patient were analyzed by the proposed non-gel sieving electrophoresis–free flow electrophoresis on the microfluidic chip. The results matched well with those detected by common clinical methods.

Golgi enrichment and proteomic analysis of developing Pinus radiata xylem by free-flow electrophoresis.

Our understanding of the contribution of Golgi proteins to cell wall and wood formation in any woody plant species is limited. Currently, little Golgi proteomics data exists for wood-forming tissues. In this study, we attempted to address this issue by generating and analyzing Golgi-enriched membrane preparations from developing xylem of compression wood from the conifer Pinus radiata. Developing xylem samples from 3-year-old pine trees were harvested for this purpose at a time of active growth and subjected to a combination of density centrifugation followed by free flow electrophoresis, a surface charge separation technique used in the enrichment of Golgi membranes. This combination of techniques was successful in achieving an approximately 200-fold increase in the activity of the Golgi marker galactan synthase and represents a significant improvement for proteomic analyses of the Golgi from conifers. A total of thirty known Golgi proteins were identified by mass spectrometry including glycosyltransferases from gene families involved in glucomannan and glucuronoxylan biosynthesis. The free flow electrophoresis fractions of enriched Golgi were highly abundant in structural proteins (actin and tubulin) indicating a role for the cytoskeleton during compression wood formation. The mass spectrometry proteomics data associated with this study have been deposited to the ProteomeXchange with identifier PXD000557.

Ion concentration polarization-based continuous separation device using electrical repulsion in the depletion region

We proposed a novel separation method, which is the first report using ion concentration polarization (ICP) to separate particles continuously. We analyzed the electrical forces that cause the repulsion of particles in the depletion region formed by ICP. Using the electrical repulsion, micro- and nano-sized particles were separated based on their electrophoretic mobilities. Because the separation of particles was performed using a strong electric field in the depletion region without the use of internal electrodes, it offers the advantages of simple, low-cost device fabrication and bubble-free operation compared with conventional continuous electrophoretic separation methods, such as miniaturizing free-flow electrophoresis (μ-FFE). This separation device is expected to be a useful tool for separating various biochemical samples, including cells, proteins, DNAs and even ions.

Tunable Membranes for Free-Flow Zone Electrophoresis in PDMS Microchip Using Guided Self-Assembly of Silica Microbeads

In this paper, we evaluate the strategy of using self-assembled microbeads to build a robust and tunable membrane for free-flow zone electrophoresis in a PDMS microfluidic chip. To fabricate a porous membrane as a salt bridge for free-flow zone electrophoresis, we used silica or polystyrene microbeads between 3-6 μm in diameter and packed them inside a microchannel. After complete evaporation, we infiltrated the porous microbead structure with a positively or negatively charged hydrogel to modify its surface charge polarity. Using this device, we demonstrated binary sorting (separation of positive and negative species at a given pH) of peptides and dyes in standard buffer systems without using sheath flows. The sample loss during sorting could be minimized by using ion selectivity of hydrogel-infiltrated microbead membranes. Our fabrication method enables building a robust membrane for pressure-driven free-flow zone electrophoresis with tunable pore size as well as surface charge polarity.

The Potential of Electrophoretic Sample Pretreatment Techniques and New Instrumentation for Bioanalysis, with A Focus on Peptidomics and Metabolomics

This Review highlights the potential of new electromigration-based sample pretreatment techniques for bioanalysis. Sample pretreatment is a challenging part of the analytical workflow, especially in the fields of peptidomics and metabolomics, where the analytes are very diverse, both in physicochemical properties and in endogenous concentration. Electromigration-based techniques have several strengths, such as fast selective analyte concentration and that complementary information on the content of a sample can be obtained when compared with more conventional (chromatography-based) techniques. In the past decade, various new electromigration-based sample pretreatment techniques have been developed, and importantly, new instrumental setups. In this Review, we provide an introduction on electromigration and its strengths. Then, selected examples of electromigration-based sample pretreatment techniques and instrumentation are discussed, namely free-flow electrophoresis, isoelectric focusing, isotachophoresis, electrodialysis, electromembrane extraction and electroextraction. Finally, the promising perspectives of electromigration-based sample pretreatment techniques are outlined.

Free‐flow electrophoresis in proteome sample preparation

An aim of proteome research is to identify the entire complement of proteins expressed in defined cell types of humans, animals, plants, and microorganisms. The approach requires searching for low abundant or even rarely expressed proteins in many cell types, as well as the determination of the protein expression levels in subcellular compartments and organelles. In recent years, rather powerful MS technologies have been developed. At this stage of MS device development, it is of highest interest to purify intact cell types or isolate subcellular compartments, where the proteins of interest are originating from, which determine the final composition of a peptide mixture. Free-flow electrophoresis proved to be useful to prepare meaningful peptide mixtures because of its improved capabilities in particle electrophoresis and the enhanced resolution in protein separation. Sample preparation by free-flow electrophoresis mediated particle separation was preferentially performed for purification of either organelles and their subspecies or major protein complexes. Especially, the introduction of isotachophoresis and interval zone electrophoresis improved the purity of the gained analytes of interest. In addition, free-flow IEF proved to be helpful, when proteins of low solubility, obtained, e.g. from cell membranes, were investigated.

A simple and highly stable free-flow electrophoresis device with thermoelectric cooling system

Complex assembly, inconvenient operations, poor control of Joule heating and leakage of solution are still fundamental issues greatly hindering application of free-flow electrophoresis (FFE) for preparative purpose in bio-separation. To address these issues, a novel FFE device was developed based on our previous work. Firstly, a new mechanical structure was designed for compact assembly of separation chamber, fast removal of air bubble, and good anti-leakage performance. Secondly, a highly efficient thermoelectric cooling system was used for dispersing Joule heating for the first time. The systemic experiments revealed the three merits: (i) 3min assembly without any liquid leakage, 80 times faster than pervious FFE device designed by us or commercial device (4h); (ii) 5s removing of air bubble in chamber, 1000-fold faster than a normal one (2h or more) and (iii) good control of Joule heating by the cooling system. These merits endowed the device high stable thermo- and hydro-dynamic flow for long-term separation even under high electric field of 63V/cm. Finally, the developed device was used for up to 8h continuous separation of 5mg/mL fuchsin acid and purification of three model proteins of phycocyanin, myoglobin and cytochrome C, demonstrating the applicability of FFE. The developed FFE device has evident significance to the studies on stem cell, cell or organelle proteomics, and protein complex as well as micro- or nano-particles.

A simple chip free‐flow electrophoresis for monosaccharide sensing via supermolecule interaction of boronic acid functionalized quencher and fluorescent dye

Here, a simple micro free-flow electrophoresis (μFFE) was developed for fluorescence sensing of monosaccharide via supermolecule interaction of synthesized boronic acid functionalized benzyl viologen (ο-BBV) and fluorescent dye. The μFFE contained two open electrode cavities and an ion-exchange membrane was sandwiched between two polymethylmethacrylate plates. The experiments demonstrated the following merits of developed μFFE: (i) up to 90.5% of voltage efficiency due to high conductivity of ion-exchange membrane; (ii) a strong ability against influence of bubble produced in two electrodes due to open design of electrode cavities; and (iii) reusable and washable separation chamber (45 mm × 17 mm × 100 μm, 77 μL) avoiding the discard of μFFE due to blockage of solute precipitation in chamber. Remarkably, the μFFE was first designed for the sensing of monosaccharide via the supermolecule interaction of synthesized ο-BBV, fluorescent dye, and monosaccharide. Under the optimized conditions, the minimum concentration of monosaccharide that could be detected was 1 × 10(-11) M. Finally, the developed device was used for the detection of 0.3 mM glucose spiked in human urine. All of the results demonstrated the feasibility of monosaccharide detection via the μFFE.