Assessing the scalability of dynamic field gradient focusing by linear modeling

Dynamic field gradient focusing (DFGF) separates and concentrates proteins in native buffers, where proteins are most soluble, using a computer-controlled electric field gradient which lets the operator adjust the pace and resolution of the separation in real-time. The work in this paper assessed whether DFGF could be scaled up from microgram analytical-scale protein loads to milligram preparative-scale loads. Linear modeling of the electric potential, protein transport, and heat transfer simulated the performance of a preparative-scale DFGF instrument. The electric potential model showed where the electrodes should be placed to optimize the shape and strength of the electric field gradient. Results from the protein transport model suggested that in 10 min the device should separate 10 mg each of two proteins whose electrophoretic mobilities differ by 5 x. Proteins with electrophoretic mobilities differing by only 5% should separate in 3 h. The heat transfer model showed that the preparative DFGF design could dissipate 1 kW of Joule heat while keeping the separation chamber at 25 degrees C. Model results pointed to DFGF successfully scaling up by 1000 x using the proposed instrument design.     

Influence of transport properties in electric field gradient focusing

Miniaturized devices for electric field gradient focusing (EFGF) were developed that consist of a cylindrical separation channel surrounded by an acrylic-based polymer hydrogel. The ionic transport properties of the hydrogel enable the manipulation of the electric field inside the separation channel. A changing cross-section design was used in which the hydrogel is shaped such that an electric field gradient is established in the separation channel. One of the challenges with this type of EFGF device has been that experimental resolution between protein analytes is lower than theoretically predicted. In order to investigate this phenomenon, a mathematical transport model was developed using FEMLAB. Model results and experimental observations showed that the reduced performance was caused by concentration gradients formed in the EFGF channel, and that these concentration gradients were the result of an imbalance in cation transport between the open separation channel and the hydrogel. Removing acidic impurities from the monomers that form the hydrogel reduced this tendency and improved the resolution. These transport-induced concentration gradients can be used to establish electric field gradients that may be useful for sample pre-concentration. Both the results of simulation and experiments demonstrate how transport-induced concentration gradients lead to the establishment of electric field gradients.     

Low-capacity channel designed for particle separation with controlled electric fields and evaluation of involved forces

Electric field is one of the suitable physical fields applicable to particle separations. Although long rectangular channel is used for particle separation in usual electrical field flow fractionation (FFF), a short low-capacity channel can replace it if the field is precisely controlled. Several separation principles are proposed with this channel. The elution behavior of particles has revealed that the gravitational, diffusion, and hydrodynamic lift force (HLF) play important roles in the determination of the elution behavior of particles. The elution threshold voltage (V(th)) was defined and experimentally determined for various system configurations and particles. The electric force no longer overcomes the other forces, and particles are taken off the wall, when the applied voltage becomes lower than V(th). V(th) values have allowed us not only to estimate surface charge density of a particle but also to evaluate the hydrodynamic lift force against particle.     

Performance optimization in electric field gradient focusing

Electric field gradient focusing (EFGF) is a technique used to simultaneously separate and concentrate biomacromolecules, such as proteins, based on the opposing forces of an electric field gradient and a hydrodynamic flow. Recently, we reported EFGF devices fabricated completely from copolymers functionalized with poly(ethylene glycol), which display excellent resistance to protein adsorption. However, the previous devices did not provide the predicted linear electric field gradient and stable current. To improve performance, Tris–HCl buffer that was previously doped in the hydrogel was replaced with a phosphate buffer containing a salt (i.e., potassium chloride, KCl) with high mobility ions. The new devices exhibited stable current, good reproducibility, and a linear electric field distribution in agreement with the shaped gradient region design due to improved ion transport in the hydrogel. The field gradient was calculated based on theory to be approximately 5.76 V/cm² for R-phycoerythrin when the applied voltage was 500 V. The effect of EFGF separation channel dimensions was also investigated; a narrower focused band was achieved in a smaller diameter channel. The relationship between the bandwidth and channel diameter is consistent with theory. Three model proteins were resolved in an EFGF channel of this design. The improved device demonstrated 14,000-fold concentration of a protein sample (from 2 ng/mL to 27 μg/mL).     

Low-voltage driven control in electrophoresis microchips by traveling electric field.

This paper presents the use of a physical model and numerical simulation in the investigation of traveling electric fields on capillary electrophoresis (CE) chips. The principal material transport mechanisms of electrokinetic migration, ionic concentration, fluid flow, and diffusion are all taken into consideration. Traditionally, the high electric field strength required for the separation of biological samples by microfluidic devices has involved the application of high external voltages. In contrast, this study presents a proposal for samples separation by means of a moving electric field within a low voltage-driven CE chip. Under this proposal, the separation channel is partitioned into a series of smaller separation zones by means of electrode pairs. This paper considers two different electrode configurations, namely arranged along a single side of the separation channel, and arranged on two sides of the separation channel. The quality of the separation achieved with these two configurations is then compared with the traditional straight separation channel approach. The results confirm that the proposed method is successful in maintaining an adequate field strength for separation purposes in a low-voltage driven CE chip. Furthermore, it is determined that the best separation results are obtained using electrodes arranged along both sides of the separation channel.     

A polarisation-independent blue-phase liquid crystal lens array using gradient electrodes

A polarisation-independent blue-phase liquid crystal lens array using gradient electrodes is proposed. A high dielectric constant layer helps to smoothen out the horizontal electric field and reduce the operating voltage. With gradient electrodes and a planar top electrode, gradient electric fields are generated and lens-like phase profile is obtained. When the applied voltage is changed, the focal length of the lens can be tuned from ∞ to 5.94 mm. Besides, the simulation results show that the lens is insensitive to polarisation while keeping parabolic-like profile.     

Numerical analysis of a dielectrophoresis field‐flow fractionation device for the separation of multiple cell types

In this study, a dielectrophoresis field‐flow fractionation device was analyzed using a numerical simulation method and the behaviors of a set of different cells were investigated. By reducing the alternating current frequency of the electrodes from the value used in the original setup configuration and increasing the number of exit channels, total discrimination in cell trajectories and subsequent separation of four cell types were achieved. Cells were differentiated based on their size and dielectric response that are represented in their real part of Clausius–Mossotti factor at different frequencies. A number of novel designs were also proposed based on the original setup configuration. It was seen that by reducing the length of the main channel and the number of electrodes at low frequencies and not changing the inlet flow velocities, cell separation was still achieved successfully, although with a slightly larger electrode voltage. The shorter main channel decreased the residence time for the cells on the chip and also reduced the overall size of the device—these were improvements over the original design. The obtained results can be used to analyze other cell types by knowing their size and dielectric properties to design geometries that can ensure separation.     

On-column conductivity detection in capillary-chip electrophoresis

On-column conductivity detection in capillary-chip electrophoresis was achieved by actively coupling the high electric field with two sensing electrodes connected to the main capillary channel through two side detection channels. The principle of this concept was demonstrated by using a glass chip with a separation channel incorporating two double-Ts. One double-T was used for sample introduction, and the other for detection. The two electrophoresis electrodes apply the high voltage and provide the current, and the two sensing electrodes connected to the separation channel through the second double-T and probe a potential difference. This potential difference is directly related to the local resistance or the conductivity of the solution defined by the two side channels on the main separation channel. A detection limit of 15 microM (600 ppb or 900 fg) was achieved for potassium ion in a 2 mM Tris-HCl buffer (pH 8.7) with a linear range of 2 orders of magnitude without any stacking. The proposed detection method avoids integrating the sensing electrodes directly within the separation channel and prevents any direct contact of the electrodes with the sample. The baseline signal can also be used for online monitoring of the electric field strength and electroosmosis mobility characterization in the separation channel.     

DC-dielectrophoretic separation of microparticles using an oil droplet obstacle

A new dielectrophoretic particle separation method is demonstrated and examined in the following experimental study. Current electrodeless dielectrophoretic (DEP) separation techniques utilize insulating solid obstacles in a DC or low-frequency AC field, while this novel method employs an oil droplet acting as an insulating hurdle between two electrodes. When particles move in a non-uniform DC field locally formed by the droplet, they are exposed to a negative DEP force linearly dependent on their volume, which allows the particle separation by size. Since the size of the droplet can be dynamically changed, the electric field gradient, and hence DEP force, becomes easily controllable and adjustable to various separation parameters. By adjusting the droplet size, particles of three different diameter sizes, 1 microm, 5.7 microm and 15.7 microm, were successfully separated in a PDMS microfluidic chip, under applied field strength in the range from 80 V cm-1 to 240 V cm-1. A very effective separation was realized at the low field strength, since the electric field gradient was proved to be a more significant parameter for particle discrimination than the applied voltage. By utilizing low strength fields and adaptable field gradient, this method can also be applied to the separation of biological samples that are generally very sensitive to high electric potential.     

Bilinear electric field gradient focusing

Electric field gradient focusing (EFGF) uses an electric field gradient and a hydrodynamic counter flow to simultaneously separate and focus charged analytes in a channel. Previously, most EFGF devices were designed to form a linear field gradient in the channel. However, the peak capacity obtained using a linear gradient is not much better than what can be obtained using conventional CE. Dynamic improvement of peak capacity in EFGF can be achieved by using a nonlinear gradient. Numerical simulation results indicate that the peak capacity in a 4-cm long channel can be increased from 20 to 150 when changing from a linear to convex bilinear gradient. To demonstrate the increased capacity experimentally, an EFGF device with convex bilinear gradient was fabricated from poly(ethylene glycol) (PEG)-functionalized acrylic copolymers. The desired gradient profile was confirmed by measuring the focusing positions of a standard protein for different counter flow rates at constant voltage. Dynamically controlled elution of analytes was demonstrated using a monolith-filled bilinear EFGF channel. By increasing the flow rate, stacked proteins that were ordered but not resolved after focusing in the steep gradient segment were moved into the shallow gradient segment, where the analyte peak resolution increased significantly. In this way, the nonlinear field gradient was used to realize a dynamic increase in the peak capacity of the EFGF method.     

Selection of the optimum electrospray voltage for gradient elution LC-MS measurements

Changes in liquid composition during gradient elution liquid chromatography (LC) coupled to mass spectrometry (MS) analyses affect the electrospray operation. To establish methodologies for judicious selection of the electrospray voltage, we monitored in real time the effect of the LC gradient on the spray current. The optimum range of the electrospray voltage decreased as the concentration of organic solvent in the eluent increased during reversed-phase LC analyses. These results and related observations provided the means to rationally select the voltage to ensure effective electrospray operation throughout gradient-elution LC separations. For analyses in which the electrospray was operated at constant voltage, a small run-to-run variation in the spray current was observed, indicating a changing electric field resulting from fouling or degradation of the emitter. Algorithms using feedback from spray current measurements that can maintain the electrospray voltage within the optimum operating range throughout gradient elution LC-MS were evaluated. The electrospray operation with voltage regulation and at a constant, judiciously selected voltage during gradient elution LC-MS measurements produced data with similar reproducibility.     

Screening and separation of charges in microscale devices: complete planar solution of the Poisson-Boltzmann equation

The Poisson-Boltzmann (PB) equation is widely used to calculate the interaction between electric potential and the distribution of charged species. In the case of a symmetrical electrolyte in planar geometry, the Gouy-Chapman (GC) solution is generally presented as the analytical solution of the PB equation. However, we demonstrate here that this GC solution assumes the presence of a bulk region with zero electric field, which is not justified in microdevices. In order to extend the range of validity, we obtain here the complete numerical solution of the planar PB equation, supported with analytical approximations. For low applied voltages, it agrees with the GC solution. Here, the electric double layers fully absorb the applied voltage such that a region appears where the electric field is screened. For higher voltages (of order 1 V in microdevices), the solution of the PB equation shows a dramatically different behavior, in that the double layers can no longer absorb the complete applied voltage. Instead, a finite field remains throughout the device that leads to complete separation of the charged species. In this higher voltage regime, the double layer characteristics are no longer described by the usual Debye parameter kappa, and the ion concentration at the electrodes is intrinsically bound (even without assuming steric interactions). In addition, we have performed measurements of the electrode polarization current on a nonaqueous model electrolyte inside a microdevice. The experimental results are fully consistent with our calculations, for the complete concentration and voltage range of interest.     

Isoelectric focusing field-flow fractionation : III. Investigation of the influence of different experimental parameters on focusing of cytochrome c in the trapezoidal cross-section channel

In addition to the electric field and pH gradient used in isoelectric focusing, a recently introduced technique, isoelectric focusing (or electrical hyperlayer) field-flow fractionation, employs the flow of the liquid carrier through a thin separation channel as a third factor affecting separation. Focusing of cytochrome c (CYTC) in a trapezoidal cross-section channel of 0.875 ml volume and 25 cm length was investigated as a function of the injection procedure, relaxation time, flow-rate of the carrier ampholyte solution and applied electric power. The influence of different initial conditions was also investigated by computer simulation. Both computed and experimental data showed an important contribution of the injection procedure and relaxation time on the retention and shape of the CYTC zone. It follows from these data that the sample should be injected as a narrow zone into the centre of the stream rather than homogeneously together with the carrier solution. For the described experimental set-up it could be demonstrated that the time necessary for zone formation should be at least 15 min and that relaxation times in excess to 20 min do not influence the final shape of the CYTC zone. It could further be shown experimentally that the sample must be injected under an applied electric field, that the relaxation time should be about 10 min, that the elution flow-rate should not be larger than 100 μl/min, that focusing becomes more efficient with increasing electric fields and that, for a given assembly and specified flow conditions, there is an electric power window only within which proper operation is possible.     

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 50×70 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.     

Towards a miniaturised system for dynamic field gradient focused separation of proteins

Separation and focusing of proteins is described in a miniaturised dynamic field gradient focusing device with a 2.5 cm x 0.1 cm channel filled with a porous polymer monolith. The separation channel is in contact with a parallel electric field channel with five individually addressable electrodes through a porous glass membrane so that a variable field can be generated that drives charged proteins electroosmotically against a constant hydrodynamic flow. Separated pre-stained proteins were detected by means of a digital camera and background subtraction.     

A blue-phase liquid crystal lens array based on dual square ring-patterned electrodes

We propose a blue-phase liquid crystal (BPLC) lens array based on dual square ring-patterned electrodes. A high dielectric constant layer is used to smoothen out the horizontal electric field and reduce the operating voltage. By creating a potential difference between the dual square ring-patterned electrodes, gradient electric fields are generated and lens-like phase profile is obtained. Besides, the focal length of the BPLC lens is adjustable with voltage changes and all simulation results indicate that the BPLC lens array is polarisation-insensitive.     

微流控芯片中超微电极的制作及其在芯片-电化学检测中的应用

设计并制作了集成有超微电极的玻璃微流控芯片.在电化学检测芯片1(EC-1)中,以光刻方法制作13μm宽的Pt超微电极,距分离管道末端30μm,优化电极体系和分离电压,检测了电泳分离的神经递质.在电化学检测芯片2(EC-2)中,制作7μm宽的超微电极,在其上游集成城墙式的膜结构,进一步腐蚀后的膜厚度为10μm,具有良好的导电性和散热性能,成功地将高压电场截至在超微电极之前,具有进一步应用于电化学检测的能力.     

Conductivity properties of carrier ampholyte pH gradients in isoelectric focusing

The conductivity properties of natural pH gradient created by carrier ampholytes were studied during the process of isoelectric focusing (IEF). IEF was performed in capillaries (10-30 mm long) or in microchips with the same channel length. A 10-30x reduction of the conductivity of the separation medium was observed during the establishment of pH gradient. Results obtained using different IEF voltages indicate that there is a nonlinear relationship between the conductivity of an established pH gradient and the applied electric field. Our theoretical analysis using a simplified model generated values that reasonably agree with the experimental data. In addition, we found that above a certain electric field ( approximately 300 V/cm), resolution does not increase with the applied voltage as predicated; we observed band-broadening and gel breakdown. The approach presented in this work can be used for optimization of the IEF separation and judicious selection of IEF conditions.     

Continuous-Flow Nanoparticle Trapping Driven by Hybrid Electrokinetics in Microfluidics

We introduce herein an efficient microfluidic approach for continuous transport and localized collection of nanoparticles via hybrid electrokinetics, which delicately combines linear and nonlinear electrokinetics driven by a composite DC-biased AC voltage signal. The proposed technique utilizes a simple geometrical structure, in which one or a series of metal strips serving as floating electrode (FE) are attached to the substrate surface and arranged in parallel between a pair of coplanar driving electrodes (DE) in a straight microchannel. On application of a DC-biased AC electric field across the channel, nanoparticles can be transported continuously by DC bulk electroosmotic flow, and then trapped selectively onto the metal strips due to AC-field induced-charge electrokinetic (ICEK) phenomenon, which behaves as counter-rotating micro-vortices around the ideally polarizable surfaces of FE. Finite-element simulation is carried out by coupling the dual-frequency electric field, flow field and sample mass transfer in sequence, for guiding a practical design of the microfluidic nanoparticle concentrator. With the optimal device geometry, the actual performance of the technique is investigated with respect to DC bias, AC voltage amplitude, and field frequency by using both latex nanospheres (∼500 nm) and BSA molecules (∼10 nm). Our experimental observation indicates nanoparticles are always enriched into a narrow bright band on the surface of each FE, and a horizontal concentration gradient even emerges in the presence of multiple metal strips, which therefore permits localized analyte enrichment. The proposed trapping method is supposed to guide an elaborate design of flexible electrokinetic frameworks embedding FE for continuous-flow analyte manipulation in modern microfluidic systems.     

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.