Free-flow Zone Electrophoresis Research Articles

Free-flow zone electrophoresis facilitated proteomics analysis of heterogeneous subpopulations in H1299 lung cancer cells

Cancer cells are usually heterogeneous, whose subpopulations contain distinct biological information that related to their unique phenotype. However, preparative separating heterogeneous cells into subpopulations is very challenging, especially based on their surface charge status. Here, free-flow zone electrophoresis (FFZE) was employed to separate and fractionate lung cells. The experimental parameters for FFZE, including voltage, running speed, and buffer pH, were optimized and the optimized method was then implemented to separate MRC-5 and H1299 cells. Normal lung cells MRC-5 could be separated into one major peak and few minor peaks. For lung cancer cell line H1299, two major peaks and a few minor peaks were fractionated. Those prepared major peaks were subjected to quantitative proteomics analysis for heterogeneous subpopulation. For H1299 cells, 2076 proteins were identified in the first major peak, and 1398 proteins were revealed in the second major peak, which increased by 13% on average (n = 3) when compared to that of before FFZE fractionation. Furthermore, we found that H1299 cells in the first major peak have higher mobility than the cells in the second one. Many cell mobility and metastasis related proteins, such as RHBDF2, TRIM59, and ADAM17, were solely expressed in the first major peak of H1299 after FFZE, indicating our method can distinguish cancer cell subpopulations with higher metastatic phenotype. Our results showed that FFZE can effectively fractionate heterogeneous cell subpopulations, which might be used for the comprehensive analysis of unique phenotypes of different cancers.

Application of Capillary and Free-Flow Zone Electrophoresis for Analysis and Purification of Antimicrobial β-Alanyl-Tyrosine from Hemolymph of Fleshfly Neobellieria bullata.

The problem of a growing resistance of bacteria and other microorganisms to conventional antibiotics gave rise to a search for new potent antimicrobial agents. Insect antimicrobial peptides (AMPs) seem to be promising novel potential anti-infective therapeutics. The dipeptide β-alanyl-tyrosine (β-Ala-Tyr) is one of the endogenous insect toxins exhibiting antibacterial activity against both Gram-negative and Gram-positive bacteria. Prior to testing its other antimicrobial activities, it has to be prepared in a pure form. In this study, we have developed a capillary zone electrophoresis (CZE) method for analysis of β-Ala-Tyr isolated from the extract of the hemolymph of larvae of the fleshfly Neobellieria bullata by reversed-phase high-performance liquid chromatography (RP-HPLC). Based on our previously described correlation between CZE and free-flow zone electrophoresis (FFZE), analytical CZE separation of β-Ala-Tyr and its admixtures have been converted into preparative purification of β-Ala-Tyr by FFZE with preparative capacity of 45.5 mg per hour. The high purity degree of the β-Ala-Tyr obtained by FFZE fractionation was confirmed by its subsequent CZE analysis.

Application of a Micro Free-Flow Electrophoresis 3D Printed Lab-on-a-Chip for Micro-Nanoparticles Analysis.

The present work describes a novel microfluidic free-flow electrophoresis device developed by applying three-dimensional (3D) printing technology to rapid prototype a low-cost chip for micro- and nanoparticle collection and analysis. Accurate reproducibility of the device design and the integration of the inlet and outlet ports with the proper tube interconnection was achieved by the additive manufacturing process. Test prints were performed to compare the glossy and the matte type of surface finish. Analyzing the surface topography of the 3D printed device, we demonstrated how the best reproducibility was obtained with the glossy device showing a 5% accuracy. The performance of the device was demonstrated by a free-flow zone electrophoresis application on micro- and nanoparticles with different dimensions, charge surfaces and fluorescent dyes by applying different separation voltages up to 55 V. Dynamic light scattering (DLS) measurements and ultraviolet−visible spectroscopy (UV−Vis) analysis were performed on particles collected at the outlets. The percentage of particles observed at each outlet was determined in order to demonstrate the capability of the micro free-flow electrophoresis (µFFE) device to work properly in dependence of the applied electric field. In conclusion, we rapid prototyped a microfluidic device by 3D printing, which ensured micro- and nanoparticle deviation and concentration in a reduced operation volume and hence suitable for biomedical as well as pharmaceutical applications.

Purification of low-abundance lysozyme in egg white via free-flow electrophoresis with gel-filtration chromatography.

As an effective separation tool, free-flow electrophoresis has not been used for purification of low-abundance protein in complex sample matrix. Herein, lysozyme in complex egg white matrix was chosen as the model protein for demonstrating the purification of low-content peptide via an FFE coupled with gel fitration chromatography (GFC). The crude lysozyme in egg while was first separated via free-flow zone electrophoresis (FFZE). After that, the fractions with lysozyme activity were condensed via lyophilization. Thereafter, the condensed fractions were further purified via a GFC of Sephadex G50. In all of the experiments, a special poly(acrylamide- co-acrylic acid) (P(AM-co-AA)) gel electrophoresis and a mass spectrometry were used for identification of lysozyme. The conditions of FFZE were optimized as follows: 130 μL/min sample flow rate, 4.9mL/min background buffer of 20mM pH 5.5 Tris-Acetic acid, 350V, and 14°C as well as 2mg/mL protein content of crude sample. It was found that the purified lysozyme had the purity of 80% and high activity as compared with its crude sample with only 1.4% content and undetectable activity. The recoveries in the first and second separative steps were 65% and 82%, respectively, and the total recovery was about 53.3%. The reasons of low recovery might be induced by diffusion of lysozyme out off P(AM-co-AA) gel and co-removing of high-abundance egg ovalbumin. All these results indicated FFE could be used as alternative tool for purification of target solute with low abundance.

Stream broadening due to fluid shear across the wider transverse dimension of a free-flow zone electrophoresis channel.

While the pressure-gradient applied along the length of a free-flow zone electrophoresis (FFZE) chamber is known to produce a parabolic flow profile for the carrier electrolyte across the narrower channel dimension (typically the channel depth), additional fluid shear can arise across the channel width due to a variety of reasons. Most commonly, any variation in the pressure-drop or channel depth across this wider dimension can lead to a gradient in the liquid flow velocity along it, significantly altering the stream broadening and, thereby, the separation performance of the assay. This article assesses the effect of such fluid shear on stream broadening during the FFZE process by describing a mathematical framework for solving the relevant advection-diffusion equation based on the method-of-moments approach. A closed-form expression for the leading order term describing the additional contribution to the spatial stream variance has been derived considering a small linear gradient in the liquid velocity across the wider transverse dimension of the FFZE chamber. The noted analysis predicts this contribution to be governed by two Péclet numbers that are evaluated based on the axial pressure-driven flow and transverse electrophoretic solute velocities. More importantly, this contribution is shown to vary quadratically with the axial distance traversed by the analyte stream as opposed to the classical linear variation known for all other stream broadening contributions in FFZE systems. The results from the analytic theory have been validated with Monte Carlo simulations, which also establish a time and length scale over which the noted analytical results are applicable.

Joule heating induced stream broadening in free-flow zone electrophoresis.

The use of an electric field in free-flow zone electrophoresis (FFZE) automatically leads to Joule heating yielding a higher temperature at the center of the separation chamber relative to that around the channel walls. For small amounts of heat generated, this thermal effect introduces a variation in the equilibrium position of the analyte molecules due to the dependence of liquid viscosity and analyte diffusivity on temperature leading to a modification in the position of the analyte stream as well as the zone width. In this article, an analytic theory is presented to quantitate such effects of Joule heating on FFZE assays in the limit of small temperature differentials across the channel gap yielding a closed form expression for the stream position and zone variance under equilibrium conditions. A method-of-moments approach is employed to develop this analytic theory, which is further validated with numerical solutions of the governing equations. Interestingly, the noted analyses predict that Joule heating can drift the location of the analyte stream either way of its equilibrium position realized in the absence of any temperature rise in the system, and also tends to reduce zone dispersion. The extent of these modifications, however, is governed by the electric field induced temperature rise and three Péclet numbers evaluated based on the axial pressure-driven flow, transverse electroosmotic and electrophoretic solute velocities in the separation chamber. Monte Carlo simulations of the FFZE system further establish a time and a length scale over which the results from the analytic theory are valid.

Comparison of antimicrobial peptide purification via free-flow electrophoresis and gel filtration chromatography.

Antimicrobial peptides (AMPs) are usually small and cationic biomolecules with broad-spectrum antimicrobial activities against pathogens. Purifying them from complex samples is essential to study their physiochemical properties. In this work, free-flow zone electrophoresis (FFZE) was utilized to purify AMPs from yeast fermentation broth. Meanwhile, gel filtration chromatography (GFC) was conducted for comparison. The separation efficiency was evaluated by SDS-PAGE analysis of the fractions from both methods. Our results demonstrated as follows: (i) FFZE had more than 30-fold higher processing capacity as compared with GFC; (ii) FFZE could achieve 87% purity and 89% recovery rate while in GFC these parameters were about 93 and 82%, respectively; (iii) the former had ∼2-fold dilution but the latter had ∼13-fold dilution. Furthermore, Tricine-SDS-PAGE, Native-PAGE, and gel IEF were carried out to characterize the purified AMPs. We found that two peptides existed as a pair with the molecularmass of ∼5.5and 7.0kDa, while the same pI 7.8. These two peptides were proved to have the antimicrobial activity through the standardized agar diffusion method. Therefore, FFZE could be used to continuously purify AMPs with high bioactivity, which will lead to its wide application in the clinical and pharmaceutical fields.

Design of suitable carrier buffer for free-flow zone electrophoresis by charge-to-mass ratio and band broadening analysis.

In this work, charge-to-mass ratio (C/M) and band broadening analyses were combined to provide better guidance for the design of free-flow zone electrophoresis carrier buffer (CB). First, the C/M analyses of hemoglobin and C-phycocyanin (C-PC) under different pH were performed by CLC Protein Workbench software. Second, band dispersion due to the initial bandwidth, diffusion, and hydrodynamic broadening were discussed, respectively. Based on the analyses of the C/M and band broadening, a better guidance for preparation of free-flow zone electrophoresis CB was obtained. Series of experiments were performed to validate the proposed method. The experimental data showed high accordance with our prediction allowing the CB to be prepared easily with our proposed method. To further evaluate this method, C-PC was purified from crude extracts of Spirulina platensis with the selected separation condition. Results showed that C-PC was well separated from other phycobiliproteins that have similar physicochemical properties, and analytical grade product with purity up to 4.5 (A620/A280) was obtained.

Enhancing resolution of free-flow zone electrophoresis via a simple sheath-flow sample injection.

In this work, a simple and novel sheath-flow sample injection method (SFSIM) is introduced to reduce the band broadening of free-flow zone electrophoresis separation in newly developed self-balance free-flow electrophoresis instrument. A needle injector was placed in the center of the separation inlet, into which the BGE and sample solution were pumped simultaneously. BGE formed sheath flow outside the sample stream, resulting in less band broadening related to hydrodynamics and electrodynamics. Hemoglobin and C-phycocyanin were successfully separated by the proposed method in contrast to the poor separation of free-flow electrophoresis with the traditional injection method without sheath flow. About 3.75 times resolution enhancement could be achieved by sheath-flow sample injection method.

An analytic description of electrodynamic dispersion in free-flow zone electrophoresis

The present work analyzes the electrodynamic dispersion of sample streams in a free-flow zone electrophoresis (FFZE) chamber resulting due to partial or complete blockage of electroosmotic flow (EOF) across the channel width by the sidewalls of the conduit. This blockage of EOF has been assumed to generate a pressure-driven backflow in the transverse direction for maintaining flow balance in the system. A parallel-plate based FFZE device with the analyte stream located far away from the channel side regions has been considered to simplify the current analysis. Applying a method-of-moments formulation, an analytic expression was derived for the variance of the sample zone at steady state as a function of its position in the separation chamber under these conditions. It has been shown that the increase in stream broadening due to the electrodynamic dispersion phenomenon is additive to the contributions from molecular diffusion and sample injection, and simply modifies the coefficient for the hydrodynamic dispersion term for a fixed lateral migration distance of the sample stream. Moreover, this dispersion mechanism can dominate the overall spatial variance of analyte zones when a significant fraction of the EOF is blocked by the channel sidewalls. The analysis also shows that analyte streams do not undergo any hydrodynamic broadening due to unwanted pressure-driven cross-flows in an FFZE chamber in the absence of a transverse electric field. The noted results have been validated using Monte Carlo simulations which further demonstrate that while the sample concentration profile at the channel outlet approaches a Gaussian distribution only in FFZE chambers substantially longer than the product of the axial pressure-driven velocity and the characteristic diffusion time in the system, the spatial variance of the exiting analyte stream is well described by the Taylor–Aris dispersion limit even in analysis ducts much shorter than this length scale.

Electrophoretic extraction of low molecular weight cationic analytes from sodium dodecyl sulfate containing sample matrices for their direct electrospray ionization mass spectrometry.

While the use of sodium dodecyl sulfate (SDS) in separation buffers allows efficient analysis of complex mixtures, its presence in the sample matrix is known to severely interfere with the mass-spectrometric characterization of analyte molecules. In this article, we report a microfluidic device that addresses this analytical challenge by enabling inline electrospray ionization mass spectrometry (ESI-MS) of low molecular weight cationic samples prepared in SDS containing matrices. The functionality of this device relies on the continuous extraction of analyte molecules into an SDS-free solvent stream based on the free-flow zone electrophoresis (FFZE) technique prior to their ESI-MS analysis. The reported extraction was accomplished in our current work in a glass channel with microelectrodes fabricated along its sidewalls to realize the desired electric field. Our experiments show that a key challenge to successfully operating such a device is to suppress the electroosmotically driven fluid circulations generated in its extraction channel that otherwise tend to vigorously mix the liquid streams flowing through this duct. A new coating medium, N-(2-triethoxysilylpropyl) formamide, recently demonstrated by our laboratory to nearly eliminate electroosmotic flow in glass microchannels was employed to address this issue. Applying this surface modifier, we were able to efficiently extract two different peptides, human angiotensin I and MRFA, individually from an SDS containing matrix using the FFZE method and detect them at concentrations down to 3.7 and 6.3 μg/mL, respectively, in samples containing as much as 10 mM SDS. Notice that in addition to greatly reducing the amount of SDS entering the MS instrument, the reported approach allows rapid solvent exchange for facilitating efficient analyte ionization desired in ESI-MS analysis.

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.

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.

Mathematical model and dynamic computer simulation on free flow zone electrophoresis

In this paper, a mathematical model was proposed for calculating physico-chemical parameters and simulating the separation process of solutes in free-flow zone electrophoresis (FFZE). Computer software was developed and implemented in a Delphi XE2 environment based on the model, which comprises zone electrophoresis, electrolyte solution, hydrodynamics and diffusion as well as conversion equations. The simulation results reveal that (i) the software is capable of simulating a dynamic process of FFZE properly; (ii) the software can simulate operation parameters (e.g., electric field, flow rate and pH value) for experimental optimization; (iii) the simulator is able to display the final electropherogram of numerous analytes in FFZE; and (iv) the electropherogram of FFZE can be automatically converted to that of capillary zone electrophoresis. In order to verify the rationality and reliability of this software, the relevant experiments were conducted and further compared with the simulations. The comparisons demonstrated the high agreement between the simulations and the experiments as well as those cited from the relevant references. In addition, effective mobility, diffusion coefficient and concentration of solutes can be conveniently gained via the software. The developed mathematical model and designed software have evident significance for the optimization of experimental conditions and basic physico-chemical parameters in FFZE.

Integrating continuous microflow reactions with subsequent micropreparative separations on a single microfluidic chip

A current challenge in continuous flow chemistry using microfluidic devices is the combination with a micropreparative separation technique for online product clean-up and analysis. Herein we describe an approach for seamless on-chip integration of continuous flow reactors with a downstream separation functionality based on free-flow electrophoresis. A prototype device was fabricated via liquid phase lithography in a one-step procedure. This proof of concept was successfully validated by performing a model reaction of amino acids with ortho-phthaldialdehyde and continuous separation of the resulting products via micro free-flow zone electrophoresis.

Analysis of Membrane-Enriched and High Molecular Weight Proteins in Leishmania infantum Promastigotes and Axenic Amastigotes

Membrane and high molecular weight (HMW) proteins tend to be underrepresented in proteome analyses. Here, we optimized a protocol designed for the extraction and purification of membranes from the protozoan parasite Leishmania using a combination of serial centrifugation and free-flow zone electrophoresis (ZE-FFE). We also enriched for Leishmania HMW proteins from total extracts using the Gelfree 8100 fractionation system. This allowed the study of expression of both membrane-enriched and HMW proteins in Leishmania infantum promastigotes and amastigotes. We identified 194 proteins with at least one transmembrane domain (TMD) and 171 HMW proteins (≥100 kDa) in the invertebrate promastigote stage and 66 proteins with at least one TMD and 154 HMW proteins in the mammalian amastigote stage. Several of the proteins identified in one of the stages are part of pathways consistent with the known biology of the parasite, with many proteins involved in lipid synthesis, numerous dynein heavy chains, and some surface antigen proteins 2 detected in the promastigote stage. Notably, some proteins involved in transport and proteolysis were detected either in promastigote or amastigote. The present study is using improved proteomic methods for studying membrane-enriched and HMW proteins helping to achieve a better understanding of the parasite life cycle.

Quantitative investigation of resolution increase of free‐flow electrophoresis via simple interval sample injection and separation

Interval free-flow zone electrophoresis (FFZE) has been used to suppress sample band broadening greatly hindering the development of free-flow electrophoresis (FFE). However, there has been still no quantitative study on the resolution increase of interval FFZE. Herein, we tried to make a comparison between bandwidths in interval FFZE and continuous one. A commercial dye with methyl green and crystal violet was well chosen to show the bandwidth. The comparative experiments were conducted under the same sample loading of the model dye (viz. 3.49, 1.75, 1.17, and 0.88 mg/h), the same running time (viz. 5, 10, 15, and 20 min), and the same flux ratio between sample and background buffer (= 10.64 × 10⁻³). Under the given conditions, the experiments demonstrated that (i) the band broadening was evidently caused by hydrodynamic factor in continuous mode, and (ii) the interval mode could clearly eliminate the hydrodynamic broadening existing in continuous mode, greatly increasing the resolution of dye separation. Finally, the interval FFZE was successfully used for the complete separation of two-model antibiotics (herein pyoluteorin and phenazine-1-carboxylic acid coexisting in fermentation broth of a new strain Pseudomonas aeruginosa M18), demonstrating the feasibility of interval FFZE mode for separation of biomolecules.

Fabrication of micro free-flow electrophoresis chip by photocurable monomer binding microfabrication technique for continuous separation of proteins and their numerical simulation

In this study, a simple, fast, and reliable method to fabricate a micro free-flow electrophoresis (μFFE) device on glass is presented. The two-dimensional depth channel in the chip was easily achieved by using a photocurable monomer (NOA 81) that served as the bonding material. In such a geometrical structure (two-dimensional depth channel), the effect of fluid behavior on the separation efficiency of micro free-flow zone electrophoresis (μFFZE) was simulated. The results of numerical simulation indicate that the pressure at the inlets may play an important role in the separation performance. Under the optimum separation conditions, four FITC-labeled amino acids were well separated, indicating the validity of the performance of the chip. Since the chip was fabricated by organic polymer bonding, it was easily recyclable through a simple re-fabrication process. The reproducibility of results from these recycling re-fabrication chips was investigated. The RSD of the resolution between FITC-L-glycine and FITC-L-phenylalanine was 5.3%. Furthermore, three FITC-labeled proteins were successfully separated with the resolution of 2.2 and 5.46, respectively, by using the coating of neutral liposome.

Protein separation using free‐flow electrophoresis microchip etched in a single step

A one-step etching method was developed to fabricate glass free-flow electrophoresis microchips with a rectangle separation microchamber (42 mm-long, 23 mm-wide and 28 microm-deep), in which two glass bridges (0.5 mm-wide) were made simultaneously to prevent bubbles formed by electrolysis near the Pt electrode from entering the separation chamber. By microchip free-flow zone electrophoresis, with 200 V voltage applied, the baseline separation of three FITC labeled proteins, ribonuclease B, myoglobin and beta-lactoglobulin, was achieved, with resolution over 1.78. Furthermore, with 2.5 mM Na(2)SO(4) added into the electrode buffer to form higher electrical field strength across separation microchamber than electrode compartments, similar resolution of samples was achieved with the applied voltage decreased to 75 V, which could obviously decrease Joule heat during continuous separation. All these results demonstrate that the free-flow electrophoresis microchip fabricated by one-step etching method is suitable for the continuous separation of proteins, which might become an effective pre-fractionation method for proteome study.

Free-Flow Zone Electrophoresis of Peptides and Proteins in PDMS Microchip for Narrow pI Range Sample Prefractionation Coupled with Mass Spectrometry

In this paper, we are evaluating the strategy of sorting peptides/proteins based on the charge to mass without resorting to ampholytes and/or isoelectric focusing, using a single- and two-step free-flow zone electrophoresis. We developed a simple fabrication method to create a salt bridge for free-flow zone electrophoresis in PDMS chips by surface printing a hydrophobic layer on a glass substrate. Since the surface-printed hydrophobic layer prevents plasma bonding between the PDMS chip and the substrate, an electrical junction gap can be created for free-flow zone electrophoresis. With this device, we demonstrated a separation of positive and negative peptides and proteins at a given pH in standard buffer systems and validated the sorting result with LC/MS. Furthermore, we coupled two sorting steps via off-chip titration and isolated peptides within specific pI ranges from sample mixtures, where the pI range was simply set by the pH values of the buffer solutions. This free-flow zone electrophoresis sorting device, with its simplicity of fabrication, and a sorting resolution of 0.5 pH unit, can potentially be a high-throughput sample fractionation tool for targeted proteomics using LC/MS.