Label-free real-time imaging in microchip free-flow electrophoresis applying high speed deep UV fluorescence scanning

We report on label-free monitoring of microfluidic free-flow electrophoresis (μFFE) separations in real-time using a custom built high speed deep UV laser scanner. In combination with a novel layout realized in fused silica (FS) FFE chips the setup was successfully applied for continuous separations and detection of unlabeled analytes including native proteins by space-resolved intrinsic deep UV fluorescence scanning.     

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 >20 min. 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.     

Unsteady transport phenomena in free-flow electrophoresis--prerequisite of ultrafast sample cleaning in microfluidic devices

The evolution partial differential equations describing the transport processes induced by hydrodynamic flow in free-flow electrophoresis (FFE) are solved by the generalized dispersion theory. Our theoretical analysis demonstrates that the central injection of solutes into a relatively fast hydrodynamic flow enables to transport them to the channel outlet well before they are spread through the width of the channel and their migration is negatively affected by a contact with walls. In this case, the axial zone spreading decreases by increasing the linear velocity of hydrodynamic flow. The resulting dependencies of convective and dispersion coefficients on the velocity of flow and parameters of the separation channel show the optimum separation conditions with respect to resolution and analysis time. Due to the unsteady character of transport processes, effective FFE separations can potentially be performed in a microfluidic device in seconds. This is a reasonable time to separate low-molecular mass impurities in the electric field. Thus, a fast and efficient sample cleaning before subsequent analysis by electrospray ionization-mass spectrometry (ESI-MS) or another separation method can be performed.     

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.     

Towards an integrated device that utilizes adherent cells in a micro-free-flow electrophoresis chip to achieve separation and biosensing

We immobilized adherent human embryonic kidney (HEK) cells—which are able to trace adenosine triphosphate (ATP) —inside a microfluidic free-flow electrophoresis (μFFE) chip in order to develop an integrated device combining separation and biosensing capabilities. HEK 293 cells loaded with fluorescent calcium indicators were used as a model system to enable the spatially and temporally resolved detection of ATP. The local position of a 20 μM ATP stream was successfully visualized by these cells during free-flow electrophoresis, demonstrating the on-line detection capability of this technique towards native, unlabeled compounds.     

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.     

Mid-scale free-flow electrophoresis with gravity-induced uniform flow of background buffer in chamber for the separation of cells and proteins

A large-scale free-flow electrophoresis (LS-FFE) is often too large for cell separation of lab scale, whereas micro-FFE (μFFE) has great difficulty in cell isolation due to easy blockage by cell accumulation in μFFE. In this study, a mid-scale FFE (MS-FFE) is developed for cell and protein separations. The volume of the separation chamber (70×40×0.1-0.8 mm) is from 280 μL to 2.24 mL, much lower than that in an LS-FFE but higher than that in a μFFE. Gravity is used for uniform flow of the background buffer only via a single pump with 16 channels and the sample is injected via an adjuster originally used for clinical intravenous injection. The experiments reveal that the hydrodynamic and electrohydrodynamic flows are much stable, and the Joule heat can be effectively dispersed without obvious positive or negative deviation as shown by the omega plots. By the device, Escherichia coli and Staphylococcus aureus, which easily accumulate to block μFFE and are separated with difficulty due to their same negative charges carried, can be well isolated under the conditions of 4.5 mM pH 8.5 Tris-boric buffer (4.5 mM Tris, 4.5 mM boric acid) with 0.10 mM ethylene diamine tetraacetic acid and 5% m/v sucrose, 200 μL/min, 800 V, and sample injection via inlet 4. The mid-scale FFE device could also be used for the separation of three model proteins of horse heart cytochrome c, myoglobin and bovine serum albumin. The device has clear significance for mid-scale separation of cells and proteins.     

Investigating peak dispersion in free-flow counterflow gradient focusing

Free-flow electrophoresis (FFE) enables the continuous separation and collection of charged solutes, and as a result, it has drawn interest as both a preparative and an analytical tool for biological applications. Recently, a free-flow counterflow gradient focusing (FF-CGF) mechanism has been proposed with the goal of improving the resolution and versatility of FFE. To realize this potential, the factors that influence solute dispersion deserve further attention, including the gradient strength and the parabolic profile of the counterflow. Therefore, the goal of this work is to develop a theoretical model to study the interplay between these factors and molecular diffusion. Overall, an asymmetric solute distribution emerges for a wide range of parameters, and this behavior can be characterized with an exponentially modified Gaussian function. Results show that FF-CGF can achieve high-resolution separations, with the potential for high-throughput protein purification. Moreover, this work provides a practical guide for optimizing experimental conditions, as well as a strong framework for understanding and developing FF-CGF further.     

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.     

Nonaqueous electrophoresis on microchips: A review

The use of organic solvents as electrolytic medium in electrophoresis has become an important alternative for the analysis of compounds that exhibit low or no solubility in water. In recent years, nonaqueous electrophoresis has been extensively explored in conventional capillary systems for different applications. On the other hand, this separation strategy is still not as popular as free solution electrophoresis on chip-based platforms due to the effects of solvent in the background electrolyte on the sample injection, detection performance, and microfluidic platform compatibility. In this way, this review summarizes the main achievements on nonaqueous microchip electrophoresis (NAME). To the best of our knowledge, this is the first review dedicated to discuss exclusively nonaqueous electrophoresis on chip-based systems. For this purpose, some important theoretical aspects involved when separations are performed in organic medium, such as equilibrium, interactions and electrophoretic considerations, are included in the review. In addition, the main challenges, advantages and influences of nonaqueous media on the sample injection, detection as well as the choice of the substrate to fabricate chip-based electrophoresis devices are highlighted. Last, examples showing the feasibility of nonaqueous microchip electrophoresis for applications exploiting different methodologies, operational, and instrumental conditions are summarized and discussed. We hope this review can be useful to spread the huge potential of nonaqueous electrophoresis on microfluidic platforms.     

Micro free-flow electrophoresis: theory and applications

Free-flow electrophoresis (FFE) is a technique that performs an electrophoretic separation on a continuous stream of analyte as it flows through a planar flow channel. The electric field is applied perpendicularly to the flow to deflect analytes laterally according to their mobility as they flow through the separation channel. Miniaturization of FFE (μFFE) over the past 15 years has allowed analytical and preparative separation of small volume samples. Advances in chip design have improved separations by reducing interference from bubbles generated by electrolysis. Mechanisms of band broadening have been examined theoretically and experimentally to improve resolution in μFFE. Separations using various modes such as zone electrophoresis, isoelectric focusing, isotachophoresis, and field-step electrophoresis have been demonstrated.

Electrophoretic separations of neuromediators on microfluidic devices

In the present work, on-chip capillary electrophoresis for the separation of neuromediators is demonstrated. The influence of separation buffer (composition, pH, SDS additive), on-chip electrokinetic sample stacking, and surface pretreatment of the PDMS-PDMS and hybrid PDMS-glass devices on the electrokinetic characteristics of microfluidics (ν_eo, μ_eo, ζ) and separation performance of on-chip capillary electrophoresis of neuromediators have been investigated. It is demonstrated that for the effective separation of neuropeptides on elastomer-based microfluidic devices, on-chip sample stacking is necessary. Field-amplified sample stacking for electroosmotic flow supported on-chip separations of neuromediators and without special design of the sample injection scheme has been demonstrated. Electrophoretic separations of fluorescently labeled analytes have been achieved within tens of seconds at injection volumes of about 110 pL, with plate numbers varying from <1000 to ∼22,000. These results demonstrate that on-chip separation methods with hybrid PDMS-glass devices are perspective for the analysis of (neuro)peptides in small volumes.     

Detection method for microchip separations

The features of analytical systems utilizing microfluidic devices, especially detection methods, are described. Electrochemical detection (EC), laser-induced fluorescence (LIF), mass spectrometry (MS), and chemical luminescence (CL) methods are covered. EC enables detection without labeling and has been used in recent years because of its low cost and sensitivity. LIF is the most generally used detection method in microchip separations. Use of LED as an excitation source for fluorescence measurement was also developed for the purpose of miniaturization of the entire system, including detection and separation. Although MS enables highly sensitive analysis, the interface between MS and micro channels is still under examination. This review with fifty-two references introduces interesting detection methods for microchip separations. Related separation methods using microfluidic devices are also discussed.     

Multistep liquid-phase lithography for fast prototyping of microfluidic free-flow-electrophoresis chips

We present a fast and versatile method to produce functional micro free-flow electrophoresis chips. Microfluidic structures were generated between two glass slides applying multistep liquid-phase lithography, omitting troublesome bonding steps or cost-intensive master structures. Utilizing a novel spacer-less approach with the photodefinable polymer polyethyleneglycol dimethacrylate (PEG-DA), microfluidic devices with hydrophilic channels of only 25 μm in height were generated. The microfluidic chips feature ion-permeable segregation walls between the electrode channels and the separation bed and hydrophilic surfaces. The performance of the chip is demonstrated by free-flow electrophoretic separation of fluorescent xanthene dyes and fluorescently labeled amino acids.     

芯片自由流电泳的研究进展

芯片自由流电泳(μ-FFE)是一种连续微制备或预分离技术,已在细胞、细胞器、蛋白质等生物样品的分析中发挥了重要作用。本文系统综述了μ-FFE的研究进展,侧重于介绍各种自由流芯片的结构、分离模式和应用。此外,还对μ-FFE的发展方向进行了展望。     

Microfluidic Pressure Driven Liquid Chromatography of Biologically Relevant Samples

An overview of the literature regarding the most recent and innovative developments in microfluidic devices for pressure-driven chromatographic separations is given, with a focus on proteomics and metabolomics applications. The applications can be considered as the main driving force for the developments in this research field, since they put high demands on the analytical technology such as for throughput, efficiency, and sensitivity and for the possibilities to interface with mass spectrometry. The developments are evaluated based on the feasibility for use in work flows for the analysis of biologically relevant samples. The literature up to the first half of 2011 is covered. Electrophoretic separations are not within the scope of this review. Several strategies have been described to obtain a retentive phase in microfluidic channels. Open channels with the stationary phase bound to the walls appear to be relatively easy to make. However, the retention in such channels is generally very low for separations of relevant samples. Microfabrication of perfectly ordered topographic structures is the most innovative of the methods discussed for the creation of stationary phases in narrow channels. Several groups work on the improvement of the surface-to-volume ratio in such channels, using different methods, and the developments towards real applications are promising. Channels packed with spherical particles and in situ polymerized monoliths for pressure-driven separations are the most frequently applied. Microfluidic devices with an integrated injection system, a (packed) separation column and a spray tip for coupling to a mass spectrometer are already commercially available, and used in practice in proteomics and metabolomics. Finally, the inherent advantages of microfluidic devices for multidimensional separations have been shown in practice in a number of studies. In these studies, pressure-driven chromatography is coupled (in series or multiplexed) to an electrophoretic separation method. The high peak capacity of such 2-dimensional separations has been shown.

     

Electrophoretic separations on chips with hydrodynamically closed separation systems

This review focuses on capillary electrophoretic separations performed on capillary electrophoresis chips (CE chips) with hydrodynamically closed separation systems in a context with transport processes (electroosmotic flow (EOF)) and hydrodynamic flow (HDF)) that may accompany the separations in these devices. It also reflects some relevant works dealing with conventional CE operating under such hydrodynamic conditions. The use of zone electrophoresis (ZE), isotachophoresis (ITP) and their on-line combination (ITP-ZE) on the single-column and column-coupling CE chips with the closed separation systems and related problems are key topics of the review. Some attention is paid to sample pretreatment in the separations performed on the CE chips. Here, mainly potentialities of the ITP-ZE combination in trace analysis applications of the miniaturized systems are discussed in a broader extent. Links between the ZE separation and detection provide a frame for the discussion of current status of the detection on the CE chips. Analytical applications illustrate potentialities of the CE chips operating with the closed separation systems (suppressed HDF and EOF) to the determination of small ions present in various matrices by ZE, ITP and ITP-ZE.     

Microfluidic chip electrophoresis for biochemical analysis

Microfluidic chip electrophoresis has been widely employed for separation of various biochemical species owing to its advantages of low sample consumption, low cost, fast analysis, high throughput, and integration capability. In this article, we reviewed the development of four different modes of microfluidics‐based electrophoresis technologies including capillary electrophoresis, gel electrophoresis, dielectrophoresis, and field (electric) flow fractionation. Coupling detection schemes on microfluidic electrophoresis platform were also reviewed such as optical, electrochemical, and mass spectrometry method. We further discussed the innovative applications of microfluidic electrophoresis for biomacromolecules (nucleic acids and proteins), biochemical small molecules (amino acids, metabolites, ions, etc.), and bioparticles (cells and pathogens) analysis. The future direction of microfluidic chip electrophoresis was predicted.     

Enhancing separation of histidine from amino acids via free-flow affinity electrophoresis with gravity-induced uniform hydrodynamic flow

In this paper, a novel mode of free-flow affinity electrophoresis (FFAE) was developed to indirectly enhance the separation of free-flow electrophoresis (FFE). In the mode of FFAE, a Ni(II) with high electric charge density and histidine (His) is chosen as a model ligand and target solute, respectively. Through the controlling of experimental conditions (10 mM pH 6.0 Na(2)HPO(4)-NaH(2)PO(4) with 2.0 mM NiCl(2)·6H(2)O background buffer), Ni(II) can combine with His and the combination leads to the high electric charge density of affinity complex of His-Ni(II) in contrast to the low density of free His molecule. But the ligand has weak interaction with uninterested amino acids. Thus, the mobility of His existing as His-Ni(II) is greatly increased from 14.5×10(-8) m(2) V(-1) s(-1) to 30.2 × 10(-8) m(2) V(-1) s(-1), while those mobilities of uninterested amino acids are almost constant. By virtue of the mode, we developed the FFAE procedure and conducted the relevant experiments. The experiments demonstrated the following merits of the FFAE technique: (i) clear enhancement of separation between the target solute of His and uninterested amino acids; (ii) simplicity, and (iii) low cost. Furthermore, the technique was used for the continuous separation of His from its complex sample, and the purity of His was near to 100%. All of the results demonstrate the feasibility of affinity separation in FFE. The developed FFAE may be used in the separation and pretreatment of some biological molecules (e.g. peptides).     

High-throughput screening and optimization approaches for chiral compounds by means of microfluidic devices

Chiral separations facilitated using microchip devices are reviewed in this paper. The first research paper on this topic was published in 1999. It was seen that analysis times are greatly reduced compared with more conventional techniques such as liquid chromatography and capillary electrophoresis, and that these devices enable the separation of chiral molecules. Method optimization can be conducted in a rather easy manner, reducing the total method development time. Finally, minute amounts of sample and buffer are used during analysis, which makes the systems ultra-economical. Although the number of applications in the chiral separation field on these miniaturized systems is still rather limited, they exhibit much potential towards high-throughput screening. Some efforts are, however, still needed regarding detection modes, because derivatisation of the samples is often needed to enable their detection.