Free-flow Electrophoresis Device Research Articles

Assessing Surface Adsorption in Cyclic Olefin Copolymer Microfluidic Devices Using Two-Dimensional Nano Liquid Chromatography-Micro Free Flow Electrophoresis Separations.

Surface interactions are a concern in microscale separations, where analyte adsorption can decrease the speed, sensitivity, and resolution otherwise achieved by miniaturization. Here, we functionally characterize the surface adsorption of hot-embossed cyclic olefin copolymer (COC) micro free-flow electrophoresis (μFFE) devices using two-dimensional nLC × μFFE separations, which introduce a 3- to 5 s plug of analyte into the device and measure temporal broadening that arises from surface interactions. COC is an attractive material for microfluidic devices, but little is known about its potential for surface adsorption in applications with continuous fluid flow and temporal measurements. Adsorption was minimal for three small molecule dyes: positively charged rhodamine 123, negatively charged fluorescein, and neutral rhodamine 110. Temporal peak widths for the three dyes ranged from 3 to 7 s and did not change significantly with increasing transit distance. Moderate adsorption was observed for Chromeo P503-labeled myoglobin and cytochrome c with temporal peak widths around 20 s. Overall, the COC surface adsorption was low compared to traditional glass devices, where peak widths are on the order of minutes. Improvements in durability, long-term performance, and ease of fabrication, combined with low overall adsorption, make the COC μFFE devices a practical choice for applications involving time-resolved continuous detection.

A review of the zone broadening contributions in free-flow electrophoresis.

The broadening of analyte streams, as they migrate through a free-flow electrophoresis (FFE) channel, often limits the resolving power of FFE separations. Under laminar flow conditions, such zonal spreading occurs due to analyte diffusion perpendicular to the direction of streamflow and variations in the lateral distance electrokinetically migrated by the analyte molecules. Although some of the factors that give rise to these contributions are inherent to the FFE method, others originate from non-idealities in the system, such as Joule heating, pressure-driven crossflows, and a difference between the electrical conductivities of the sample stream and background electrolyte. The injection process can further increase the stream width in FFE separations but normally influencing all analyte zones to an equal extent. Recently, several experimental and theoretical works have been reported that thoroughly investigate the various contributions to stream variance in an FFE device for better understanding, and potentially minimizing their magnitudes. In this review article, we carefully examine the findings from these studies and discuss areas in which more work is needed to advance our comprehension of the zone broadening contributions in FFE assays.

Separation and purification of active ingredients in tobacco by free-flow electrophoresis.

The active ingredients from tobacco extracts were continuously separated and purified using a homemade free-flow electrophoresis apparatus. A rectangular free flow electrophoresis device was constructed for the continuous separation and preparation, and the operating conditions of the device were optimized. The fractions obtained from the free-flowing component collection unit were then detected by HPLC and GC-MS. The results showed that a 90% methanol-water solution could maximize the extraction of the active components from tobacco. Chlorogenic acid and nicotine were enriched in three and four of 24 fractions, respectively, after free-flow isoelectric focusing electrophoresis. 2-Hydroxy-2-cyclopentene-1-one, 1-(2-methyl-1,3-oxathiolan-2-yl) ethanone, nornicotine, cotinine, and scopolamine were separated and enriched synchronously. Overall, the use of free-flow electrophoresis technology for the separation and purification of the active substances in tobacco can improve the comprehensive utilization rate of tobacco.

Fluorescence Imaging Characterization of the Separation Process in a Monolithic Microfluidic Free-Flow Electrophoresis Device Fabricated Using Low-Temperature Co-Fired Ceramics.

A monolithic microfluidic free-flow electrophoresis device, fabricated using low-temperature co-fired ceramic technology, is presented. The device integrates gold electrodes and a 20 µm thick transparent ceramic optical window, suitable for fluorescence imaging, into a multilevel microfluidic chamber design. The microfluidic chamber consists of a 60 µm deep separation chamber and two, 50 µm deep electrode chambers separated by 10 µm deep side channel arrays. Fluorescence imaging was used for in-chip, spatial-temporal characterization of local pH variations in separation conditions as well as to characterize the separation process. The device allowed baseline resolution separation of a sample mixture of Fluorescein, Rhodamine 6G, and 4-Methylumbelliferone at pH 7.0, in only 6 s, using 378 V.s/cm. The results demonstrate the possibility of studying a chemical process using fluorescence imaging within the traditional fields of low-temperature co-fired ceramics technology, such as high-electrical-field applications, while using a simple fabrication procedure suitable for low-cost mass production.

Multi-channel contactless conductivity detection device for online detection of free-flow electrophoresis separation

现有自由流电泳(FFE)装置因不具备在线检测功能,其实用性仍然存在明显不足。针对这一问题,该工作发展了一种多通道电容耦合式非接触电导检测(MC-C4D)装置并开发了自动测量软件。MC-C4D装置采用了并行分时的非接触电导检测技术,即由多个同样的非接触电导检测模块并行排列,而单个电导检测模块又由多个非接触电导检测池组成,采用模拟开关切换这些检测池,能够分时检测流经相应检测池溶液的电导率。多个电导检测模块的检测池总数等于FFE的组分数,它们分别串行接入到FFE各流路中,这样MC-C4D装置就可在线并行分时在线测量各组分溶液的电导率。为验证所设计MC-C4D装置的检测性能,采用配制的氯化钾标准溶液作为检测对象对MC-C4D装置进行了标定和测试。实验数据表明,MC-C4D装置电导率检测范围为0.015~2.5 mS/cm,检出限(LOD)为0.002 mS/cm,日内相对标准偏差(RSD, n=3)为2.31%,测量相对误差(RE)为3.03%和通道间测量相对偏差为1.60%,这些参数表明该装置检测范围较大,LOD低,重复性好,准确性高,通道间测量相对偏差小。另外,将MC-C 4D装置应用于往复式自由流等电聚焦电泳(RFFIEF)在蛋白质聚焦过程中对各组分溶液电导率进行实时在线检测,结果表明,所开发的MC-C4D装置不仅可实现对FFE各组分溶液电导率的实时在线检测,而且还可在RFFIEF实验中辅助掌握分离的实验进度,提高FFE装置的实用性。

Continuous separation of submicron-scale oil droplets in aqueous electrolyte by electrophoretic migration

Separation of tiny oil droplets, which is not readily achieved by conventional oily wastewater treatment, is becoming important in the petrochemical industries with regard to public health and the global environment. Herein, we report a continuous and filterless separation of submicron-scale oil droplets based on electrophoretic migration in a three-dimensional (3D) printed microfluidic free-flow electrophoresis (μFFE) device which is miniaturized system of conventional electrodialysis (ED). Microscale electrodialysis system with commercial ion exchange membrane (IEM) is proposed to control the dynamics of negatively (or positively) charged tiny oil droplets. We examined separation performance for various parameters, such as applied voltage, recovery ratio, flow velocity, solution concentration, and flow-channel dimension, with visualization of fluorescent oil droplets. Separation of several non-fluorescent submicron-scale oil droplets (e.g. gasoline engine, diesel engine, gear, and palm oils) were also investigated, including measurement of droplet diameters and surface charge (zeta potential). Future scale-up of the proposed microfluidic ED system will be a promising method for the treatment and relevant applications of industrial oily wastewater.

Reduced surface adsorption in 3D printed acrylonitrile butadiene styrene micro free-flow electrophoresis devices.

We have 3D printed and fabricated micro free-flow electrophoresis (µFFE) devices in acrylonitrile butadiene styrene (ABS) that exhibit minimal surface adsorption without requiring additional surface coatings or specialized buffer additives. 2D, nano LC-micro free flow electrophoresis (2D nLC × µFFE) separations were used to assess both spatial and temporal broadening as peaks eluted through the separation channel. Minimal broadening due to wall adsorption was observed in either the spatial or temporal dimensions during separations of rhodamine 110, rhodamine 123, and fluorescein. Surface adsorption was observed in separations of Chromeo P503 labeled myoglobin and cytochrome c but was significantly reduced compared to previously reported glass devices. Peak widths of <30 s were observed for both proteins. For comparison, Chromeo P503 labeled myoglobin and cytochrome c adsorb strongly to the surface of glass µFFE devices resulting in peak widths >20min. A 2D nLC × µFFE separation of a Chromeo P503 labeled tryptic digest of BSA was performed to demonstrate the high peak capacity possible due to the low surface adsorption in the 3D printed ABS devices, even in the absence of surface coatings or buffer additives.

Continuous purification of reaction products by micro free-flow electrophoresis enabled by large area deep-UV fluorescence imaging.

Microreactors have gained increasing attention in their application toward continuous micro flow synthesis. An unsolved problem of continuous flow synthesis is the lack of techniques for continuous product purification. Herein, we present a micro free-flow electrophoresis device and accompanying setup that enables the continuous separation and purification of unlabeled organic synthesis products. The system is applied to the separation and purification of triarylmethanes. For imaging of the unlabeled analytes on-chip a novel setup for large area (3.6 cm2) deep ultra violet excitation fluorescence detection was developed. Suitable separation conditions based on low conductivity electrophoresis buffers were devised to purify the product. With the optimized conditions, starting materials and product of the synthesis were well separated (R > 1.2). The separation was found to be very stable with relative standard deviations of the peak positions smaller than 3.5% over 15 min. The stable conditions enabled collection of the separated compounds, and purity of the product fraction was confirmed using capillary electrophoresis and mass spectrometry. This result demonstrates the great potential of free-flow electrophoresis as a technique for product purification or continuous clean-up in flow synthesis. Graphical Abstract Micro free-flow electrophoresis (μFFE) allows continuous separation and purification of small organic synthesis products. Enabled by a novel deep-UV imaging setup starting materials and product of a recently developed synthesis for triarylmethanes could be purified. Thereby demonstrating the potential of μFFE as continuous purification technique for micro flow synthesis.

3D Printed Micro Free-Flow Electrophoresis Device.

The cost, time, and restrictions on creative flexibility associated with current fabrication methods present significant challenges in the development and application of microfluidic devices. Additive manufacturing, also referred to as three-dimensional (3D) printing, provides many advantages over existing methods. With 3D printing, devices can be made in a cost-effective manner with the ability to rapidly prototype new designs. We have fabricated a micro free-flow electrophoresis (μFFE) device using a low-cost, consumer-grade 3D printer. Test prints were performed to determine the minimum feature sizes that could be reproducibly produced using 3D printing fabrication. Microfluidic ridges could be fabricated with dimensions as small as 20 μm high × 640 μm wide. Minimum valley dimensions were 30 μm wide × 130 μm wide. An acetone vapor bath was used to smooth acrylonitrile-butadiene-styrene (ABS) surfaces and facilitate bonding of fully enclosed channels. The surfaces of the 3D-printed features were profiled and compared to a similar device fabricated in a glass substrate. Stable stream profiles were obtained in a 3D-printed μFFE device. Separations of fluorescent dyes in the 3D-printed device and its glass counterpart were comparable. A μFFE separation of myoglobin and cytochrome c was also demonstrated on a 3D-printed device. Limits of detection for rhodamine 110 were determined to be 2 and 0.3 nM for the 3D-printed and glass devices, respectively.

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.

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.

Fabrication and Examination of a Novel Triangular-shape Free Flow Electrophoresis Device

A novel triangular-shape FFE device was designed based on free flow electrophoresis(FFE) theory.In comparison with traditional FFE device,the shape of separation chamber of the novel device was triangular.The sample was driven forward by pump to form laminar flow when applied single-tube sample injection without the background buffer.The structure of the device was studied as well as the electric field distribution theory.The mixture of methylene blue and neutral red was successfully separated on this device when voltage was 400 V and flow rate was 0.5 mL/min.Traditional rectangle-shape device could reduce the copper and zinc contents of Shangdong noble metallic solution to 51.46% and 58.87%,respectively,when U=70 V and v=1.0 mL/min.The contents of copper and zinc could be reduced to 52.77% and 53.94% after the disposal of triangular-shape device when U=70 V,v=1.0 mL/min and third and fourth electrode chambers were in series with 500 Ω and 1000 Ω,respectively.Thus,the triangular-shape device could reach the similar separation effect with traditional rectangle-shape device.Therefore,this device might be promising in the field of consecutive separation of macroscale samples.

Simply enhancing throughput of free‐flow electrophoresis via organic‐aqueous environment for purification of weak polarity solute of phenazine‐1‐carboxylic acid in fermentation of Pseudomonas sp. M18

Herein, a simple novel free-flow electrophoresis (FFE) method was developed via introduction of organic solvent into the electrolyte system, increasing the solute solubility and throughput of the sample. As a proof of concept, phenazine-1-carboxylic acid (PCA) from Pseudomonas sp. M18 was selected as a model solute for the demonstration on feasibility of novel FFE method on account of its faint solubility in aqueous circumstance. In the developed method, the organic solvent was added into not only the sample buffer to improve the solubility of the solute, but also the background buffer to construct a uniform aqueous-organic circumstance. These factors of organic solvent percentage and types as well as pH value of background buffer were investigated for the purification of PCA in the FFE device via CE. The experiments revealed that the percentage and the types of organic solvent exerted major influence on the purification of PCA. Under the optimized conditions (30 mM phosphate buffer in 60:40 (v/v) water-methanol at an apparent pH 7.0, 3.26 mL/min background flux, 10-min residence time of injected sample, and 400 V), PCA could be continuously purified from its impurities. The flux of sample injection was 10.05 μL/min, and the recovery was up to 93.7%. An 11.9-fold improvement of throughput was found with a carrier buffer containing 40% (v/v) methanol, compared with the pure aqueous phase. The developed procedure is of evident significance for the purification of weak polarity solute via FFE.

Miniaturised free flow isotachophoresis of bacteria using an injection moulded separation device

A new design of miniaturised free flow electrophoresis device has been produced. The design contains a separation chamber that is 45mm long by 31.7mm wide with a depth of 50μm and has nine inlet and nine outlet holes to allow for fraction collection. The devices were formed of polystyrene with carbon fibre loaded polystyrene drive electrodes and produced using injection moulding. This means that the devices are low cost and can potentially be mass produced. The devices were used for free flow isotachophoresis (FFITP), a technique that can be used for focussing and concentrating analytes contained within complex sample matrices. The operation of the devices was demonstrated by performing separations of dyes and bacterial samples. Analysis of the output from FFITP separations of samples containing the bacterium Erwinia herbicola, a biological pathogen, by cell culturing and counting showed that fractionation of the output was achieved.

Isolation of DNA aptamers using micro free flow electrophoresis

A micro free flow electrophoresis (μFFE) device was used to select DNA aptamers for human immunoglobulin E (IgE). The continuous nature of μFFE allowed 1.8 × 10(14) sequences to be introduced over a period of 30 min, a 300-fold improvement in library size over capillary electrophoresis based selections (CE-SELEX). Four rounds of selection were performed within four days. Aptamers with low nM dissociation constants for IgE were identified after a single round of μFFE selection.

PDMS free-flow electrophoresis chips with integrated partitioning bars for bubble segregation

In this work, a microfluidic free-flow electrophoresis device with a novel approach for preventing gas bubbles from entering the separation area is presented. This is achieved by integrating partitioning bars to reduce the channel depth between electrode channels and separation chamber in order to obtain electrical contact and simultaneously prevent bubbles from entering the separation area. The three-layer sandwich chip features a reusable carrier plate with integrated ports for fluidic connection combined with a softlithographically cast microfluidic PDMS layer and a sealing glass slide. This design allows for a straightforward and rapid chip prototyping process. The performance of the device is demonstrated by free-flow zone electrophoretic separations of fluorescent dye mixtures as well as by the separation of labeled amines and amino acids with separation voltages up to 297 V.

Controlling of band width, resolution and sample loading by injection system in a simple preparative free-flow electrophoresis with gratis gravity

In this paper, the controllable band width, resolution and sample loading were investigated by the injection system of free-flow electrophoresis (FFE) with gratis gravity. Two general injection methods were described herein. The first method was the one in which sample injection fluxes were variable with constant background flux, while the second was the one in which the background fluxes were flexible with stable sample flux. With methyl green and crystal violet as two viewable model compounds, a series of experiments were performed, and the experimental results revealed that (1) the sample band width could be under desiring control through the regulation of ratios between sample and background fluxes, (2) the separative resolution could be also adjusted elaborately via the regulation of flux ratios during the separation of methyl green and crystal violet with only one charge disparity, and (3) the sample loading could be conveniently controlled via the flux ratios and an approximate maximum sample loading could be selected under the condition of just completed separation of two adjoining solutes. In addition, it was observed that the flux ratio had soft influence on the separative resolution of two solutes. These results were of significance to the designs on band width, resolution and sample loading in the newly developed FFE device with gratis gravity.

Separation of proteins using a novel two-depth miniaturized free-flow electrophoresis device with multiple outlet fractionation channels

A novel free-flow electrophoresis glass chip design with two-depth etched structures for the separation and fractionation of proteins is presented. The microfluidic structures etched in two depths enhance the flow characteristics inside the miniaturized device. A novel nine-port outlet interface enables the fractionation of the separated analytes. The separation and focussing of a protein sample mixture demonstrated the ability of the new chip.

Synchronized, Continuous-Flow Zone Electrophoresis

A new method for performing continuous electrophoretic separation of complex mixtures in microscale devices is proposed. Unlike in free-flow electrophoresis devices, no mechanical pumping is required--both fluid transport and separation are driven electrokinetically. This gives the method great potential for on-a-chip integration in multistep analytical systems. The method enables us to collect fractionated sample and tenfold purification is possible. The model of the operation is presented and a detailed description of the optimal conditions for performing purification is given. The chip devices with 10-microm-deep separation chamber of 1.5 mm x 4 mm in size were fabricated in glass. A standard microchip electrophoresis setup was used. Continuous separation of rhodamine B, rhodamine 6G, and fluorescein was accomplished. Purification was demonstrated on a mixture containing rhodamine B and fluorescein, and the recovery of both fractions was achieved. The results show the feasibility of the method.

Bubble-Free Operation of a Microfluidic Free-Flow Electrophoresis Chip with Integrated Pt Electrodes

In order to ensure a stable and efficient separation in microfluidic free-flow electrophoresis (FFE) devices, various methods and chips have been presented until now. A major concern hereby is the generation of gas bubbles caused by electrolysis and the resulting disturbances in the position of the separated analyte lanes. Instable lane positions would lead to a decreased resolution in sample collection over time which certainly would be problematic when incorporating a stationary detector system. In contrast to our previous publications, in which we implemented laborious semipermeable membranes to keep bubbles outside the separation region, here we describe an electrochemical approach to suppress the electrolysis of water molecules and therefore bubble formation. This approach allowed a simpler and additionally a closed chip device with integrated platinum electrodes. With the use of this chip, the successful separation of three fluorescent compounds was demonstrated. Quinhydrone, which is a complex of hydroquinone and p-benzoquinone, was added only to the local flow streams along the electrodes, preventing mixing with the separation media and sample. The electrical current was generated via the oxidization and reduction of hydroquinone and p-benzoquinone up to a certain limit of the electrical current without gas formation. The separation stability was investigated for the chip with and without quinhydrone, and the results clearly indicated the improvement. In contrast to the device operating without quinhydrone, a 2.5-fold increase in resolution was achieved. Furthermore, separation was demonstrated within tens of milliseconds. This chemical approach with its high miniaturization possibilities offers an interesting alternative, in particular for low-current miniaturized FFE systems, in which large and open electrode reservoirs are not tolerable.