Free-flow Isoelectric Focusing Research Articles

Switchable pH actuators and 3D integrated salt bridges as new strategies for reconfigurable microfluidic free-flow electrophoretic separation

We present novel strategies for reconfigurable, high-throughput microfluidic free-flow electrophoretic separation using electrically switchable pH actuators and 3D integrated salt bridges to allow rapid formation of stable pH gradients and efficient electrophoresis. The pH actuator is achieved by microfluidic integration of bipolar membranes which change electrolyte pH by injecting excess H(+) or OH(-) ions produced by a field-enhanced water dissociation phenomenon at the membrane junction upon voltage bias. The technique does not require conventional multiple buffer inflows and leaves no gas production as experienced in electrolysis, thus providing stable pH gradients for isoelectric focusing (IEF) separation. With the pH actuator inactivated, the platform can perform zone electrophoretic (ZE) separation in a medium of constant pH. We also describe the use of 3D integrated ion conductive polymers that serve as salt bridges for improving the voltage efficiency of electrophoresis and to allow high throughput. The proof of concept was successfully demonstrated for free-flow IEF and ZE separation of protein mixtures showing the potential and the simplicity of the platform for high-throughput and high-precision sample separation.

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.

Interaction of Proteins Identified in Human Thyroid Cells

Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains.

Microfluidic free-flow electrophoresis chips with an integrated fluorescent sensor layer for real time pH imaging in isoelectric focusing

Functional microfluidic free-flow electrophoresis chips with integrated fluorescent pH sensors are presented. Polyethylene glycol based structures were fabricated that allowed for integration of both functions on a single microchip. Microchips were applied in free-flow isoelectric focusing of model compounds and proteins with on-line monitoring of pH during microscale electrophoretic separation.

Inkjet Printed Structures for Smart Lab-on-Chip Systems

Lab-on-Chip microfluidic systems require onboard functionalities such as fluid pumping, heating and fluorescent or chemical sensing. An approach is presented to realize these structures by inkjet printing of functional materials or material sandwiches and subsequent curing or sintering. In detail an electro-active polymer membrane pump with passive microstructured valves, an open loop controlled ohmic heater structure for maintaining discrete fluid temperatures and electrodes for free-flow isoelectric focusing along with fluorescent hydrogel pads for pH sensing are discussed that all together are printed onto the cover foil of a polymer based microfluidic chip platform at the size of a microscope slide.

Metabolic enzyme diversity in different human thyroid cell lines and their sensitivity to gravitational forces

Many cancer cells show unique protein expression patterns. We used proteome technology including MS, free flow isoelectric focusing and Western blotting to determine current concentrations of metabolic enzymes in healthy and malignant human thyroid cells. Three different types of human thyroid cells were investigated after they had been cultured under equal conditions. MS revealed high quantities of glycolytic enzymes and moderate quantities of citric acid cycle enzymes in malignant FTC-133 cells with abnormal LDH B-chains, high quantities of glycolytic enzymes and marginal quantities of citric acid cycle enzymes in normal HTU-5 cells, and low quantities of glycolytic enzymes and marginal quantities of citrate cycle enzymes in malignant CGTH-W1 cells with abnormal LDH A-chains. When an alteration of gene expression activity was challenged physically by removing gravity forces, the concentrations of various glycolytic enzymes were changed in normal and malignant thyroid cells. However, the changes varied among the different cell types. Different cellular alignment of the enzymes could be one reason for the cell type-specific behavior as demonstrated by histological analysis of alpha-enolase.

Enantiomeric resolution of tryptophan via stereoselective binding in an ion-exchange membrane partitioned free flow isoelectric focusing system

The optical resolution efficiency of a membrane system that integrates stereoselective affinity dialysis and ion-exchange membrane partitioned free flow isoelectric focusing (FFIEF) is shown to be superior to normal affinity dialysis (AD) and affinity ultrafiltration (AUF) membrane processes under similar experimental conditions, i.e. by using the same sulfonated polyetherketone (SPEK) membranes and identical human serum albumin (HSA) to tryptophan ratio of 0.75. The chiral separation is achieved by isolating the unbound tryptophan, which contains D-tryptophan in excess, and the protein–tryptophan complex into the permeation and feed chambers, respectively, by controlling their migration under an external electric field. The separation factor is increased with increasing protein concentration while the permeation flux can be enhanced by increasing the operating current. The rationale for using HSA instead of BSA as the chiral selector, and the use of four-chamber system rather than two are also discussed.

Generation of a miniaturized free-flow electrophoresis chip based on a multi-lamination technique—isoelectric focusing of proteins and a single-stranded DNA fragment

Free-flow electrophoresis techniques have been applied for separations in various areas of chemistry and biochemistry. Here we focus on the generation of a free-flow electrophoresis chip and direct monitoring of the separation of different molecules in the separation bed of the miniaturized chip. We demonstrate a fast and efficient way to generate a low-cost micro-free-flow electrophoresis (μFFE) chip with a filling capacity of 9.5 μL based on a multi-lamination technique. Separating webs realized by two transfer-adhesive tapes avoid the problem of gas bubbles entering the separation area. The chip is characterized by isoelectric focusing markers (IEF markers). The functionality of the chip is demonstrated by free-flow isoelectric focusing (FFIEF) of the proteins BSA (bovine serum albumin) and avidin and a single-stranded DNA (ssDNA) fragment in the pH range 3 to 10. The separation voltage ranges between 167 V cm(-1) and 422 V cm(-1), depending on the application.

A proteomic approach to analysing spheroid formation of two human thyroid cell lines cultured on a random positioning machine

The human cell lines FTC-133 and CGTH W-1, both derived from patients with thyroid cancer, assemble to form different types of spheroids when cultured on a random positioning machine. In order to obtain a possible explanation for their distinguishable aggregation behaviour under equal culturing conditions, we evaluated a proteomic analysis emphasising cytoskeletal and membrane-associated proteins. For this analysis, we treated the cells by ultrasound, which freed up some of the proteins into the supernatant but left some attached to the cell fragments. Both types of proteins were further separated by free-flow IEF and SDS gel electrophoresis until their identity was determined by MS. The MS data revealed differences between the two cell lines with regard to various structural proteins such as vimentin, tubulins and actin. Interestingly, integrin α-5 chains, myosin-10 and filamin B were only found in FTC-133 cells, while collagen was only detected in CGTH W-1 cells. These analyses suggest that FTC-133 cells express surface proteins that bind fibronectin, strengthening the three-dimensional cell cohesion.

Microfluidic preparative free‐flow isoelectric focusing in a triangular channel: System development and characterization

A preparative scale free-flow IEF device is developed and characterized with the aim of addressing needs of molecular biologists working with protein samples on the milligrams and milliliters scale. A triangular-shape separation channel facilitates the establishment of the pH gradient with a corresponding increase in separation efficiency and decrease in focusing time compared with that in a regular rectangular channel. Functionalized, ion-permeable poly(acrylamide) gel membranes are sandwiched between PDMS and glass layers to both isolate the electrode buffers from the central separation channel and also to selectively adjust the voltage efficiency across the separation channel to achieve high electric field separation. The 50 x 70 mm device is fabricated by soft lithography and has 24 outlets evenly spaced across a pH gradient between pH 4 and 10. This preparative free-flow IEF system is investigated and optimized for both aqueous and denaturing conditions with respect to the electric field and potential efficiency and with consideration of Joule-heating removal. Energy distribution across the functionalized polyacrylamide gel is investigated and controlled to adjust the potential efficiency between 15 and 80% across the triangular separation channel. The device is able to achieve constant electric fields high as 370+/-20 V/cm through the entire triangular channel given the separation voltage of 1800 V, enabling separation of five fluorescent pI markers as a demonstration example.

Chemical modification of P84 polyimide as anion-exchange membranes in a free-flow isoelectric focusing system for protein separation

By means of amination with diamine and methylation with methyl iodide, we have modified P84 microporous polyimide membranes with characteristics of highly charged anion-exchange membranes. FTIR (Fourier transform infrared-attenuated total reflection) and XPS (X-ray photoelectron spectroscopy) confirm the amination and methylation reactions on membrane surfaces. The intrinsic properties of the newly developed anion-exchange membranes have been fully characterized in terms of ζ-potentials, electrical resistances, PWPs (pure water permeation) and pore size distributions. By using the newly developed membranes, a free-flow isoelectric focusing (IEM-FFIEF) has been set up for the separation of myoglobin (Mb) and lysozyme (Lys) mixtures. Experimental data show that (1) the Mb flux via the highly charged P84 anion-exchange membrane can be 10 times higher than that of the original P84 membrane and (2) the high surface charge is the predominant factor for the enhanced Mb flux. HPLC (high performance liquid chromatography) results show that not only the Mb flux was high, but also its purity in the permeate side is extremely high. It is therefore concluded that the diamine and methyl iodide modifications can effectively modulate P84 nano-porous membranes with anion-exchange characteristics, which is suitable for the IEM-FFIEF application.

Application of free‐flow IEF to identify protein candidates changing under microgravity conditions

Using antibody-related methods, we recently found that human thyroid cells express various proteins differently depending on whether they are cultured under normal gravity (1g) or simulated microgravity (s-microg). In this study, we performed proteome analysis in order to identify more gravity-sensitive thyroid proteins. Cells cultured under 1g or s-microg conditions were sonicated. Proteins released into the supernatant and those remaining in the cell fragments were fractionated by free-flow IEF. The fractions obtained were further separated by SDS-gel electrophoresis. Selected gel pieces were excised and their proteins were determined by MS. A total of 235 different proteins were found. Out of 235 proteins, 37 appeared to be first identifications in human thyroid cells. Comparing SDS gel lanes of equally numbered free-flow IEF fractions revealed similar patterns with a number of identical bands if proteins of a distinct cell line had been applied, irrespective of whether the cells had been cultured under 1g or s-microg. Most of the identical band pairs contained identical proteins. However, the concentrations of some types of proteins were different within the two pieces of gel. Proteins that concentrated differently in such pieces of gel are considered as candidates for further investigations of gravitational sensitivity.

Synthesis of mosaic membranes and application for egg white protein fractionation by partitioned free-flow isoelectric focusing (FFIEF)

In this article, multi-component protein fractionation was studied by mosaic membrane partitioned free-flow isoelectric focusing (FFIEF). The mosaic membranes were molecularly designed through a blending of aminated poly(2,6-dimethyl-1,4-phenylene oxide) (APPO) and sulfonated polysulfone (SPSf) with polysulfone (PSf). All cast membranes were prepared from the same dope but phase inverted in different coagulants to produce different morphologies. Membrane characterizations through field emission scanning electron microscopy (FESEM) imaging, streaming potential, pore size distribution and PWP (pure water permeation) demonstrated that the self-prepared membranes possessed a variety of membrane structures and charge characteristics. These mosaic membranes were applied in FFIEF as the selective barriers. Experimental results show that a blend of APPO and SPSf with PSf is an effective route to produce highly charged mosaic membranes, which can be used in the separation of five pure protein components from chicken egg white using the mosaic-membrane partitioned FFIEF.

Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation

Isoelectric focusing (IEF) is the first step for two-dimensional (2D) gel electrophoresis and plays an important role in sample purification for proteomics. However, biases in protein size and pI resolution, as well as limitations in sample volume, gel capacity, sample loss, and experimental time, remain challenges. In order to address some of the limitations of traditional IEF, we present a microfluidic free flow IEF (FF-IEF) device for continuous protein separation into 24 fractions. The device reproducibly establishes a nearly linear pH gradient from 4 to 10. Optimized dynamic coatings of 4% poly(vinyl alcohol) (PVA) minimize peak broadening by transverse electrokinetic flows. Even though the device operates at high electric fields (up to 370 V/cm), efficient cooling maintains solution temperature inside the separation channel controllably in the range 2-25 degrees C. Protein samples with a dynamic concentration range from microg/mL to mg/mL can be loaded into the microdevice at a flow rate of 1 mL/h and residence time of approximately 12 min. By using a protein complex of nine proteins and 13 isoforms, we demonstrate improved separation with the FF-IEF system over traditional 2D gel electrophoresis. Device-to-device reproducibility is also illustrated through the efficient depletion of the albumin and hemoglobin assays. Post-device sample concentrations result in a 10-20-fold increase, which allow for isolation and detection of low abundance proteins. The separation of specific proteins from a whole cell lysate is demonstrated as an example. The microdevice has the further benefits of retaining high molecular weight proteins, providing higher yield of protein that has a broader range in pI, and reducing experimental time compared to conventional IEF IGP gel strip approaches.

Self-sharpening phenomenon arisen by ion-exchange membranes in multi-compartment free-flow isoelectric focusing (IEM-FFIEF)

Abstract In this article, the self-sharpening phenomenon arisen by ion-exchange membranes is studied. In order to reduce the overlapped components in a single chamber, aminated poly(2,6-dimethyl-1,4-phenylene oxide) (APPO) based anion-exchange membranes are applied in free-flow isoelectric focusing (FFIEF) instead of conventional immobiline membranes as the selective mass transfer media. The APPO polymers with different amination rates are blended with polysulfone and cast on non-woven clothes by the phase inversion technology. Characterizations of XPS scanning, streaming potential and ion-exchange capacity (IEC) demonstrate that the self-prepared membranes posses different extent of amination and IEC values. The performances of the three prepared APPO membranes with different IEC values are compared. Nine pieces identical solid phase porous APPO membranes are employed in FFIEF instead of pH imbedded gel-like immobiline membrane with a protein mixture comprising bovine serum albumin, myoglobin and lysozyme as a separation model. Experimental results show that membranes with the higher charge density perform not only the higher mass transfer rate, but also the stronger “self-sharpening” function. Therefore, the highly charged porous membranes are favorable in reducing components’ overlaps in individual chamber for multi-component protein separations.

Microchip free flow isoelectric focusing for protein prefractionation using monolith with immobilized pH gradient

Microchip free flow IEF (muFFIEF) with monolithic IPG was proposed for protein prefractionation. The monolithic materials were first prepared by UV irradiation in a microchamber of 43 mm length, 23 mm width, and 38 mum depth. Carrier ampholytes (CAs) were further immobilized on the monolith by chemical bonding, to form a stable pH gradient. By such a technique, the continuous introduction of CAs in traditional muFFIEF could be avoided, not only to decrease the operation cost, but also to avoid the interference of CAs for the further protein identification by MS/MS. With a fluorescence microscope as the detector, under the optimal conditions, the separation of FITC labeled beta-lactoglobulin and carbonic anhydrase, with 0.9 unit difference on pI, was achieved with good reproducibility. The experimental results demonstrated that under the experimental conditions we applied, the separation mechanism of muFFIEF with M-IPG materials might be the cooperative effects of IEF and CZE, and the former one plays a predominant role.

Free-flow isoelectric focusing microfluidic device with glass coating by sol–gel methods

A device for microfluidic free-flow isoelectric focusing (μ-FFIEF) was fabricated in a glass-coated PDMS structure. The microfluidic channel was made by a PDMS replica molding and standard soft photolithography technique. For the prefractionation and preconcentration of a protein mixture, we patterned palladium electrodes on the glass substrate and coated with TEOS–sol to enhance the bonding with PDMS. The PDMS microchannel was coated with glass by flushing TEOS–sol and annealing at 100 °C. The optimum focusing condition was obtained by pH indicator with ampholyte solution made with MES. Model proteins, RFP and EGFP, were successfully concentrated around 214 μm and 357 μm from the anode, respectively. The optimum focusing condition in the μ-FFIEF device was an electric potential of 1.5 V/cm and pH 6.0. The focusing time in the channel without glass coating was approximately 30 s, but the breakdown of the electrode by electrolysis started immediately when the electric field was applied. However, the glass-coated device was kept intact under continuous application of voltage giving a stable pH gradient, but with twice the focusing time.

Free flow and capillary isoelectric focusing of bacteria from the tomatoes plant tissues

The means of the preconcentration and preseparation of selected species or pathovars of bacteria directly from the plant tissue suspension by free flow isoelectric focusing are introduced here. After the focusing, the resulting fraction of microorganisms, native or dynamically modified by the non-ionogenic tenside on the basis of pyrenebutanoate, was separated by capillary isoelectric focusing and/or cultivated and positively identified by gas chromatographic analysis of fatty acid methyl esters. Simultaneously, capillary isoelectric focusing with UV and fluorometric detection was used for the rapid estimation of unknown isoelectric points of the examined plant pathogenic species of genus Clavibacter, Xanthomonas and Pseudomonas prior to the preconcentration and preseparation. The microorganisms were of different origin, native and/or dynamically modified by the non-ionogenic tenside.

Miniaturizing free‐flow electrophoresis – a critical review

Free-flow electrophoresis (FFE) separation methods have been developed and investigated for around 50 years and have been applied not only to many types of analytes for various biomedical applications, but also for the separation of inorganic and organic substances. Its continuous sample preparation and mild separation conditions make it also interesting for online monitoring and detection applications. Since 1994 several microfluidic, miniaturized FFE devices were developed and experimentally characterized. In contrast to their large-scale counterparts microfluidic FFE (mu-FFE) devices offer new possibilities due to the very rapid separations within several seconds or below and the requirement for sample volumes in the microliter range. Eventually, these mu-FFE systems might find application in so-called lab-on-a-chip devices for real-time monitoring and separation applications. This review gives detailed information on the results so far published on mu-FFE chips, comprising its four main modes, namely free-flow zone electrophoresis (FFZE), free-flow IEF (FFIEF), free-flow ITP (FFITP), and free-flow field-step electrophoresis (FFFSE). The principles of the different FFE modes and the basic underlying theory are given and discussed with special emphasis on miniaturization. Different designs as well as fabrication methods and applied materials are discussed and evaluated. Furthermore, the separation results shown indicate that similar separation quality with respect to conventional FFE systems, as defined by the resolution and peak capacity, can be achieved with mu-FFE separations when applying much lower electrical voltages. Furthermore, innovations still occur and several approaches for hyphenated, more integrated systems have been proposed so far, some of which are discussed here. This review is intended as an introduction and early compendium for research and development within this field.

High-performance protein separation by ion exchange membrane partitioned free-flow isoelectric focusing system

The objective of this study is to investigate a combination of ion exchange membranes and free-flow isoelectric focusing (FFIEF) technology for high-selectivity and high-flux protein separation, in which ion exchange membranes are used as the separation media. An FFIEF device has been designed and extensive experiments have been conducted to prove its effectiveness in enhancing the protein separation performance. Three types of membranes were employed in this work and they were commercial microfiltration (MF) ion exchange membranes, commercial neutral UF cellulose membranes, and home-made ultrafiltration sulfonated polysulfone (UF SPSf) ion exchange membranes. The protein separation results show that the home-made UF SPSf membranes have the superior selectivity and flux to other membranes. This is due to the fact that a stable pH gradient across the membranes as well as the interaction between the protein molecules and membrane surface plays an important role in the high-performance protein separation. By applying a semi-batch separation process and optimizing various experimental conditions, a high-purity ( > 90 % ) and concentrated target protein is obtained at the permeation side of the home-made UF SPSf membranes with a high flux.