On-line preconcentration of protein in capillary electrophoresis with an end-column cellulose acetate-based porous membrane

A simple on-line preconcentration method of protein for capillary electrophoresis (CE) using a cellulose acetate (CA)-coated porous membrane was proposed. CA membrane is fabricated at one of the ends of the column that allows the passage of buffer ions but excludes larger protein molecules. Protein sample is continuously electrokinetically loaded and trapped by the membrane. When injection is completed, the direction of the electric field is switched and the trapped proteins are then separated by conventional CE procedure. The results achieved showed that the preconcentration mechanism of this method was based on size-exclusion effect. Bovine serum albumin (BSA) was used for model protein sample, and signal enhancement of 550-fold with 15 min injection time was achieved.     

Sweeping: concentration mechanism and applications to high-sensitivity analysis in capillary electrophoresis

Sweeping in capillary electrophoresis (CE) involves the interaction of a pseudostationary phase (PS) in the separation solution and a sample in the matrix that is free of the PS used. The PS includes not only the PSs employed in electrokinetic chromatography, but also complexation reagents such as borate. The sample matrix could have a lower, similar, or higher conductance than the separation solution. Thus, the basic condition for sweeping is a sample matrix free of the additive. The accumulation of analyte molecules during the interaction makes this interesting phenomenon very useful as an on-line preconcentration method for CE. Preconcentration occurs due to chromatographic partitioning, complexation, or any interaction between analytes and PS. Contact between analyte and PS is facilitated by the action of electrophoresis and is independent of electroosmosis. The analyte, PS, or both should have electrophoretic velocities when an electric field is applied. The extent of preconcentration is dictated by the strength of the interaction involved. From tens to several thousand-fold improvements in detector response for many neutral and charged analytes have been achieved with this technique, suggesting sweeping as a general approach to on-line preconcentration in CE. The mechanism and applications of the sweeping phenomenon under different experimental conditions are discussed in this review, with particular emphasis on a better understanding of the sweeping mechanism under reduced electric field (high conductivity) in the sample zone.     

自由溶液和筛分介质条件下芯片DNA瞬间等速电泳预浓缩的比较

利用芯片电泳方法考察瞬间等速电泳-筛分电泳偶联分析的结果,比较了自由溶液和筛分介质中DNA瞬间等速电泳的预浓缩效果.结果显示,相比较于筛分介质条件,自由溶液瞬间等速电泳有利于改善预浓缩和后续筛分电泳分离效果.对此结果的解释是:自由溶液条件下DNA迁移速度的提高可以延长瞬间等速电泳持续时间,有利于提高预浓缩效率.此外,样品压缩区带在自由溶液-筛分介质界面的二次富集也是预浓缩效果得到改善的原因之一.     

Electrokinetic transport through nanochannels

This article presents a numerical study of the electrokinetic transport phenomena (electroosmosis and electrophoresis) in a three-dimensional nanochannel with a circular cross-section. Due to the nanometer dimensions, the Boltzmann distribution of the ions is not valid in the nanochannels. Therefore, the conventional theories of electrokinetic flow through the microchannels such as Poisson-Boltzmann equation and Helmholtz-Smoluchowski slip velocity approach are no longer applicable. In the current study, a set of coupled partial differential equations including Poisson-Nernst-Plank equation, Navier-Stokes, and continuity equations is solved to find the electric potential field, ionic concentration field, and the velocity field in the three-dimensional nanochannel. The effects of surface electric charge and the radius of nanochannel on the electric potential, liquid flow, and ionic transport are investigated. Unlike the microchannels, the electric potential field, ionic concentration field, and velocity field are strongly size-dependent in nanochannels. The electric potential gradient along the nanochannel also depends on the surface electric charge of the nanochannel. More counter ions than the coions are transported through the nanochannel. The ionic concentration enrichment at the entrance and the exit of the nanochannel is completely evident from the simulation results. The study also shows that the flow velocity in the nanochannel is higher when the surface electric charge is stronger or the radius of the nanochannel is larger.     

The role of external electric fields in enhancing ion mobility, drift velocity, and drift-diffusion rates in aqueous electrolyte solutions

Molecular simulations have been carried out using the method of molecular dynamics to investigate the role of external electric fields on the ion mobility, drift velocity, and drift-diffusion rate of ions in aqueous electrolyte solutions. These properties are critical for a range of processes including electrodialysis, electro-deionization, electrophoresis, and electroosmosis. Our results show that external electric fields relax the hydrated ion structure at significantly larger time scales (between 300 and 800 ps), than most other relaxation processes in solutions (generally of the order of 1 ps). Previous studies that did not account for the much longer relaxation times did not observe this behavior for ions even with very high electric fields. External electric fields must also overcome several (at least two or more) activation energy barriers to significantly change the structure of hydrated ions. As a result, the dynamic behavior changes almost in bands as a function of electric field strengths, rather than linearly. Finally, the effect of the field is much less dramatic on water than the ions. Thus electric fields will be of more significance in processes that involve the transport of ions (such as electro-deionization) than the transport of water (electroosmosis).     

Rapid and efficient isotachophoretic preconcentration in free solution coupled with gel electrophoresis separation on a microchip using a negative pressure sampling technique

We present a novel isotachophoresis–gel electrophoresis (ITP–GE) microchip system designed for rapid and efficient isotachophoretic preconcentration coupled with gel electrophoresis separation by using a negative pressure sampling technique. The overall ITP–GE procedure involves only three steps: sample loading, ITP preconcentration and GE separation and was controlled by a simple and compact negative pressure sampling device, which is composed of a vacuum vessel, a three-way electromagnetic valve and a single high voltage power supply. During the sample loading stage, a negative pressure was applied via a three-way electromagnetic valve in headspace of the two sealed sample waste reservoirs (SWs). A sandwiched sample zone between a leading and a terminating electrolyte zone was formed in the channel intersection in less than 1 s. Once the three-way electromagnetic valve was switched to connect SWs to ambient atmosphere to release vacuum in SWs, ITP preconcentration in free solution and GE separation in the 4% hydroxyethylcellulose (HEC) sieving material were consequently activated under the electric potentials applied. The performance of present approach was evaluated by using DNA fragments as model analytes. Compared to conventional cross microchip GE using electrokinetic pinched injection, an average signal enhancement of 185-fold was obtained with satisfactory resolution. The results demonstrated the ITP–GE approach possessing an exciting potential of high sensitivity and short sampling time with significant simplification in operation and instrumentation.     

Recent progress of on-line sample preconcentration techniques in microchip electrophoresis

This review highlights recent developments and applications of on-line sample preconcentration techniques to enhance the detection sensitivity in microchip electrophoresis (MCE); references are mainly from 2008 and later. Among various developed techniques, we focus on the sample preconcentration based on the changes in the migration velocity of analytes in two or three discontinuous solutions system, since they can provide the sensitivity enhancement with relatively easy experimental procedures and short analysis times. The characteristic features of the on-line sample preconcentration applied to microchip electrophoresis (MCE) are presented, categorized on the basis of "field strength-" or "chemically" induced changes in the migration velocity. The preconcentration techniques utilizing field strength-induced changes in the velocity include field-amplified sample stacking, isotachophoresis and transient-isotachophoresis, whereas those based on chemically induced changes in the velocity are sweeping, transient-trapping and dynamic pH junction.     

Microchip electrophoresis of DNA following preconcentration at photopatterned gel membranes

Rapid separation of nucleic acids by microchip electrophoresis could streamline many biological applications, but conventional chip injection strategies offer limited sample stacking, and thus limited sensitivity of detection. We demonstrate the use of photopatterned polyacrylamide membranes in a glass microfluidic device, with or without fixed negative charges, for preconcentration of double-stranded DNA prior to electrophoretic separation to enhance detection limits. We compared performance of the two membrane formulations (neutral or negatively charged) as a function of DNA fragment size, preconcentration time, and preconcentration field strength, with the intent of optimizing preconcentration performance without degrading the subsequent electrophoretic separation. Little size-dependent bias was observed for either membrane formulation when concentrating dsDNA > 100 bp in length, while the negatively charged membrane more effectively blocks passage of single-stranded oligonucleotide DNA (20-mer ssDNA). Baseline resolution of a six-band dye-labeled ladder with fragments 100-2000 bp in size was obtained in <120 s of separation time, with peak efficiencies in the range of 2000-15,000 plates/cm, and detection limits as low as 1 pM per single dye-labeled fragment. The degree of preconcentration is tunable by at least 49-fold, although the efficiency of preconcentration was found to have diminishing returns at high field and/or long times. The neutral membrane was found to be more robust than the negatively charged membrane, with approximately 2.5-fold larger peak area during the subsequent separation, and less decrease in resolution upon increasing the preconcentration field strength.     

Numerical simulation of electrokinetic injection techniques in capillary electrophoresis microchips

The effective design and control of a capillary electrophoresis (CE) microchip requires a thorough understanding of the electrokinetic transport phenomena associated with its microfluidic injection system. The present study utilizes a numerical simulation approach to investigate these electrokinetic transport processes and to study the control parameters of the injection process. Injection systems with a variety of different configurations are designed and tested, including the cross-form, T-form, double-T-form, variable-volume focused flow cross-form, and variable-volume triple-T-form configuration. Each injection system cycles through a predetermined series of steps in which the magnitudes and distributions of the applied electric field are precisely manipulated in order to effectuate a virtual valve. This study investigates the sample leakage effect associated with each of the injection configurations and applies the double-L, pullback, and focusing injection techniques to minimize the sample leakage effect. The injection methods presented in this paper have the exciting potential for use in high-quality, high-throughput chemical analysis applications and throughout the micro-total-analysis systems field.     

High-sensitivity capillary and microchip electrophoresis using electrokinetic supercharging

Electrokinetic supercharging (EKS) is considered as one of the most powerful online preconcentration techniques in electrophoresis. It combines the efficient preconcentration power of field-amplified sample injection and the exceptional selective nature of transient isotachophoresis. It has a wide range of applications to different types of analytes ranging from small ions to large proteins and DNA fragments. This comprehensive review--up to date--provides listing for all the works, developments, and advances in EKS. The review will pay particular attention to innovations, new methodologies for manipulation, challenges for improving the detection sensitivity, and various applications of EKS in capillaries and microchips.     

High-resolution DNA separation in microcapillary electrophoresis chips utilizing double-L injection techniques

An experimental and numerical investigation into the use of high-resolution injection techniques to separate DNA fragments within electrophoresis microchips is presented. The principal material transport mechanisms of electrokinetic migration, fluid flow, and diffusion are considered, and several variable-volume injection methods are discussed. A detailed analysis is provided of a double-L injection technique, which employs appropriate electrokinetic manipulations to reduce sample leakage within the microchip. The leakage effect in electroosmotic flow (EOF) is investigated using a sample composed of rhodamine B and Cy3 dye. Meanwhile, the effects of sample leakage in capillary electrophoresis (CE) separation are studied by considering the separation of 100-base pairs (bp) DNA ladders and HaeIII-digested PhiX-174 DNA samples. The present experimental and simulation results indicate that the unique injection system employed in the current microfluidic chip has the ability to replicate the functions of both the conventional cross-channel and the shift-channel injection systems. Furthermore, applying the double-L injection method to these two injection systems is shown to reduce sample leakage significantly. The proposed microfluidic chip and double-L injection technique developed in this study have an exciting potential for use in high-resolution, high-throughput biochemical analysis applications and in many other applications throughout the micrototal analysis systems field.     

Simulation of electrophoretic separations by the flux-corrected transport method

Electrophoretic separations at typical experimental electric field strengths have been simulated by applying the flux-corrected transport (FCT) finite difference method to the transient, one-dimensional electrophoresis model. The performance of FCT on simulations of zone electrophoresis (ZE), isotachophoresis (ITP), and isoelectric focusing (IEF) has been evaluated. An FCT algorithm, with a three-point, central spatial discretization, yields numerical solutions without numerical oscillations or spurious peaks, which have plagued previously-published second-order solutions to benchmark ZE and ITP problems. Moreover, the FCT technique captures sharp zone boundaries and IEF peaks more accurately than previously-published, first-order upwind schemes.     

线性聚丙烯酰胺凝胶毛细管电泳的迁移特性

使用线性聚丙烯酰胺作为筛分介质,对片段长度为80～584bp的标准DNA样品进行毛细管电泳,利用激光诱导荧光方法检测信号,荧光染料为溴化乙啶。改变电场强度100～375V/cm,得到的迁移率曲线与电场强度和DNA片段长度成复杂的函数关系,已有的经典理论模型:Ogston模型、Reptation无拉伸模型和Reptation拉伸模型都不能正确地描述实验观察到的迁移率随电场强度和DNA片段长度的变化情况。因此,提出一种修正的Ogston筛分理论,假定迁移的DNA分子在电场强度方向延展拉伸,如同小分子穿过凝胶筛孔。在该修正模型中,DNA的迁移率仅依赖于电场强度、筛分介质浓度和片段长度,很好地解释了实验现象。     

Plasma Parameters around a Chain-Like Structure of Dust Particles in an External Electric Field

The formation of a 1D chain-like structure of dust particles in a low-temperature argon plasma was studied. A new numerical model for calculation of the self-consistent spatial distribution of plasma parameters around a chain of dust particles was presented. The model described the motion of positively charged ions in the electric potential of several negatively charged dust particles, taking into account the action of an external electric field. The main advantage of the model was that the charges of the dust particles and the interparticle distances were determined self-consistently. As a result of numerical simulations, the dependencies of the spatial distributions of the plasma parameters (the densities of electrons and ions and the self-consistent electric potential) near the dust particles chain on the strength of the external electric field, an external force acted on the last particle, and the mean free path of the ions was determined. The obtained results made it possible to describe the process of the formation of chain-like structures of dust particles in discharge plasma.     

Micellar electrokinetic chromatography on microchips

This review highlights the methodological and instrumental developments in microchip micellar EKC (MCMEKC) from 1995. The combination of higher separation efficiencies in micellar EKC (MEKC) with high-speed separation in microchip electrophoresis (MCE) should provide high-throughput and high-performance analytical systems. The chip-based separation technique has received considerable attention due to its integration ability without any connector. This advantage allows the development of a multidimensional separation system. Several types of 2-D separation microchips are described in the review. Since complicated channel configurations can easily be fabricated on planar substrates, various sample manipulations can be carried out prior to MCMEKC separations. For example, mixing for on-chip reactions, on-line sample preconcentration, on-chip assay, etc., have been integrated on MEKC microchips. The application of on-line sample preconcentration to MCMEKC can provide not only sensitivity enhancement but also the elucidation of the preconcentration mechanism due to the visualization ability of MCE. The characteristics of these sample manipulations on MEKC microchips are presented in this review. The scope of applications in MCMEKC covers mainly biogenic compounds such as amino acids, peptides, proteins, biogenic amines, DNA, and oestrogens. This review provides a comprehensive table listing the applications in MCMEKC in relation to detection methods.     

Over 1000-fold protein preconcentration for microliter-volume samples at a pH junction using capillary electrophoresis

An effective protein preconcentration technique specifically designed for microliter-volume samples is presented. The preconcentration is based on the capturing of protein in its isoelectric point (pI) within an applied electric field, using a pH junction created by a discontinuous buffer system. The buffers were chosen to selectively preconcentrate proteins of neutral pI, myoglobin in this case, while removing other proteins with acidic or basic pIs. For the suppression of electro-osmotic flow (EOF) and protein adsorption, the capillary inner wall was modified with a zwitterionic phospholipid bilayer coating. A preconcentration factor of up to 1700 was obtained for a 1 microg/mL solution of myoglobin. The preconcentration was completed in approximately 20 min. Several sample introduction conditions were presented to accommodate sample volume from one to a few hundreds of microliters. The final volume of the preconcentrated sample band was estimated to be approximately 5 nL.     

Electrophoresis of diffuse soft particles

A theory is presented for the electrophoresis of diffuse soft particles in a steady dc electric field. The particles investigated consist of an uncharged impenetrable core and a charged diffuse polyelectrolytic shell, which is to some extent permeable to ions and solvent molecules. The diffuse character of the shell is defined by a gradual distribution of the density of polymer segments in the interspatial region separating the core from the bulk electrolyte solution. The hydrodynamic impact of the polymer chains on the electrophoretic motion of the particle is accounted for by a distribution of Stokes resistance centers. The numerical treatment of the electrostatics includes the possibility of partial dissociation of the hydrodynamically immobile ionogenic groups distributed throughout the shell as well as specific interaction between those sites with ions from the background electrolyte other than charge-determining ions. Electrophoretic mobilities are computed on the basis of an original numerical scheme allowing rigorous evaluation of the governing transport and electrostatic equations derived following the strategy reported by Ohshima, albeit within the restricted context of a discontinuous chain distribution. Attention is particularly paid to the influence of the type of distribution adopted on the electrophoretic mobility of the particle as a function of its size, charge, degree of permeability, and solution composition. The results are systematically compared with those obtained with a discontinuous representation of the interface. The theory constitutes a basis for interpreting electrophoretic mobilities of heterogeneous systems such as environmental or biological colloids or swollen/deswollen microgel particles.     

Theoretical investigation on the performance of DNA electrophoresis under programmed step electric field strength: Two‐step condition

Programmed step electric field strength is a simple‐to‐use technique that has already been reported to be effective to enhance the efficiency or speed of DNA electrophoresis. However, a global understanding and the details of this technique are still vague. In this paper, we investigated the influence of programmed step electric field strength by theoretical calculation and concentrated on a basic format named as two‐step electric field strength. Both subtypes of two‐step electric field strength conditions were considered. The important parameters, such as peak spacing, peak width, resolution, and migration time, were calculated in theory to understand the performance of DNA electrophoresis under programmed step electric field strength. The influence of two‐step electric field strength on DNA electrophoresis was clearly revealed on a diagram of resolution versus migration time. Both resolution and speed of DNA electrophoresis under two‐step electric field strength conditions are simply expressed by the shape of curves in the diagram. The possible shapes of curve were explored by calculation and shown in this paper. The subtype II of two‐step electric field strength brings drastic variation on the resolution. Its limitations of enhancement and deterioration of resolution were predicted in theory.     

Fast DNA sequencing up to 1,000 bases by capillary electrophoresis using poly(N,N-dimethylacrylamide) as a separation medium

Poly(N,N-dimethylacrylamide) (PDMA) with a molecular mass of 5.2 x 10(6) g/mol has been synthesized and used in DNA sequencing analysis by capillary electrophoresis (CE). A systematic investigation is presented on the effects of different separation conditions, such as injection amount, capillary inner diameter, polymer concentration, effective separation length, electric field and temperature, on the resolution. DNA sequencing up to 800 bases with a resolution (R) limit of 0.5 (and 1,000 bases with a resolution limit of 0.3) and a migration time of 96 min was achieved by using 2.5% w/v polymer, 150 V/cm separation electric field, and 60 cm effective separation length at room temperature on a DNA sample prepared with FAM-labeled--21M13 forward primer on pGEM3Zf(+) and terminated with ddCTP. Ultrafast and fast DNA sequencing up to 420 and 590 bases (R > or = 0.5) were also achieved by using 3% w/v polymer and 40 cm effective separation length with a separation electric field of 525 and 300 V/cm, and a migration time of 12.5 and 31.5 min, respectively. PDMA has low viscosity, long shelf life and dynamic coating ability to the glass surface. The unique properties of PDMA make it a very good candidate as a separation medium for large-scale DNA sequencing by capillary array electrophoresis (CAE).     

Experimental and numerical investigation into leakage effect in injectors of microfluidic devices

This paper performs an experimental and numerical investigation into low-leakage injectors designed for electrophoresis microchips. The principal material transport mechanisms of electrokinetic migration, fluid flow, and diffusion are considered in developing a mathematical model of the electrophoresis process. Low-leakage injectors designed with injection channels orientated at various included angles are designed and tested. The numerical and experimental results indicate that the injector with a 30 degrees included angle successfully minimizes sample leakage and has an exciting potential for use in high-quality, high-throughput chemical analysis procedures and in many other applications in the field of micro-total analysis systems.