基于微芯片电泳的脱氧核糖核酸片段的浓缩和分离

采用超负荷电动供给(electrokinetic supercharging, EKS)预浓缩技术,在微芯片电泳(MCE)上对脱氧核糖核酸（DNA）片段进行浓缩和分离。EKS是集合样品电动进样(EKI)和过渡等速电泳(tITP)的一种在线浓缩方法。研究表明：采用该方法后,在40.5 mm长的单通道芯片上能够实现对低浓度样品的大量进样、浓缩和基线分离。在普通的紫外检测条件(检测波长为260 nm)下,对DNA片段的平均检出限(S/N=3)约为0.07 mg/L,仅为十字芯片上的微芯片电泳检出限的1/40。本文还对浓缩过程中的一些关键因素和定性分析进行了探讨。     

Optimization of the electrokinetic supercharging preconcentration for high-sensitivity microchip gel electrophoresis on a cross-geometry microchip

We developed a novel on-line preconcentration procedure for microchip gel electrophoresis (MCGE), which enables application of electrokinetic supercharging (EKS) for highly sensitive detection of DNA fragments on a cross-geometry microchip. In comparison with conventional pinched injection using the cross microchip, the present approach allows loading a much larger amount of the sample by taking advantage of a newly developed operational mode. In order to obtain high preconcentration effect and prevent splitting of an enriched sample into subchannels, i.e., off the detector range, effects of the voltage applied on the reservoirs and the time of isotachophoretic preconcentration were examined. The optimal balance between the voltage and time was found for a high-sensitivity analysis of DNA fragments. After experimental optimization the detection limit of a 150 bp fragment was as low as 0.22 mg/L (S/N = 3) that is 10 times better than using the conventional pinched injection.     

High-sensitivity capillary and microchip electrophoresis using electrokinetic supercharging preconcentration. Insight into the stacking mechanism via computer modeling

This review discusses recent progress in the application of one of the most effective in-line preconcentration techniques used in electrophoresis in capillaries and microchips, electrokinetic supercharging (EKS). Conventionally considered as a transient isotachophoresis (tITP) step put into effect after the electrokinetic sample injection (EKI), EKS presumes that the electrolyte filled into the capillary (or microchip channel) comprises a co-ion acting as a leading ion to stack the injected analytes. Subsequently, to create the tITP state, one needs an additional injection of a suitable terminating ion. As a resulting increase in sensitivity strongly depends on the performance of both EKS stages, two theoretical sections are focused on hints for proper arrangement of EKI and tITP elaborated by means of computer simulation. In particular, factors affecting the injected amount of analytes, different modes of introducing the sample, suitable combinations of leading and terminating ions, and optimization of supporting electrolyte compositions are discussed with an objective to increase the enrichment factors. A comprehensive coverage of recent EKS applications in capillary and microchip electrophoresis, including metal ions, pharmaceuticals, peptides, DNA fragments, and proteins, demonstrates attainable sensitivity enhancements up to two orders of magnitude. This should make this method exportable to other analytes and facilitate its more widespread use to applications that require low limits of detection.     

Study of a novel sample injection method (floating electrokinetic supercharging) for high-performance microchip electrophoresis of DNA fragments

Aiming to achieve high-performance analysis of DNA fragments using microchip electrophoresis, we developed a novel sample injection method, which was given the name of floating electrokinetic supercharging (FEKS). In the method, electrokinetic injection (EKI) and ITP preconcentration of samples was performed in a separation channel, connecting two reservoir ports (P3 and P4) on a cross-geometry microchip. At these two stages, side channels, crossing the separation channel, and their ports (P1 and P2) were electrically floated. After the ITP-stacked zones passed the cross-part, they were eluted for detection by using leading ions from P1 and P2 that enabled electrophoresis mode changing rapidly from ITP to zone electrophoresis (ZE). Possible sample leakage at the cross-part toward P1 and P2 was studied in detail on the basis of computer simulation using a CFD-ACE+ software and real experiments, through which it was validated that the analyte recovery to the separation channel was almost complete. The FEKS method successfully contributed to higher resolution and shorter analysis time of DNA fragments on the cross-microchip owing to more rapid switching from ITP status to ZE separation in comparison with our previous EKS procedure realized on a single-channel microchip. Without any degradation of resolution, the achieved LODs were on average ten times better than using conventional pinched injection.     

High-sensitive capillary zone electrophoresis analysis by electrokinetic injection with transient isotachophoretic preconcentration: electrokinetic supercharging

The principle of an on-line preconcentration method for capillary zone electrophoresis (CZE) named electrokinetic supercharging (EKS), is described and based on computer simulation the preconcentration behavior of the method is discussed. EKS is an electrokinetic injection method with transient isotachophoretic process, is a powerful preconcentration technique for the analysis of dilute samples. After filling the separation capillary with supporting electrolyte, an appropriate amount of a leading electrolyte was filled and the electrokinetic injection was started. After a while, terminating electrolyte was filled subsequently and migration current was applied. This procedure enabled the introduction of a large amount of sample components from a dilute sample without deteriorating separation. Computer simulation of the electrokinetic injection revealed that EKS was effective for the preconcentration of analytes with wide mobility ranges by proper choice of transient isotachophoresis (ITP) system and electroosmotic flow (EOF) should be suppressed to increase injectable amount of analytes under constant voltage mode. A test mixture of rare-earth chlorides was used to demonstrate the uses of EKS-CZE. When a 100 microL sample was used, the low limit of detectable concentration was 0.3 microg/L (1.8 nM for Er), which was comparable or even better than that of ion chromatography and inductively coupled plasma-atomic emission spectrometry (ICP-AES).     

Electrokinetic supercharging preconcentration and microchip gel electrophoretic separation of sodium dodecyl sulfate-protein complexes

A microchip gel electrophoresis (MCGE) method with electrokinetic supercharging (EKS, electrokinetic injection with transient isotachophoresis) on a single channel chip was developed for high-sensitive detection of a standard mixture of six proteins (phosphorylase b, albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor, and alpha-lactalbumin) in the form of sodium dodecyl sulfate (SDS) complexes. An average lower limit of detectable concentration (LLDC) achieved using UV detection at 214 nm was 0.27 microg/mL that is 30 times lower than that of conventional MCGE on a cross geometry chip. The calibration curves for molecular weight and concentration of SDS-protein complexes suggested that the present EKS-MCGE method had a better linear dynamic range and benefited future applications for qualitative and quantitative analysis of unknown protein samples. It was found that an excessive amount of unbound SDS in the sample deteriorated the preconcentration effect and resolution. The developed method appears greatly promising for high-speed and high sensitive analysis of SDS-proteins by MCGE.     

Electrokinetic supercharging preconcentration prior to CGE analysis of DNA: Sensitivity depends on buffer viscosity and electrode configuration

Aiming to high sensitivity DNA analysis by CGE, electrokinetic supercharging (EKS) approach was adopted in this article. EKS is known as an online preconcentration technique that combines electrokinetic sample injection (EKI) with transient ITP (tITP). Herein, two factors of buffer viscosity and electrode configuration were studied to further improve EKS performance. An ultralow‐viscosity Tris‐Boric acid‐EDTA (TBE) buffer solution, consisted of 2% low‐molecular‐weight hydroxypropyl methyl cellulose (HPMC) and 6% mannitol and with pH 8.0 adjusted by boric acid, was applied. The boric acid would make a complex with mannitol and generates borate polyanion, which acts as the leading ion for tITP process. The new electrode configuration, a Pt ring around capillary, was modified on Agilent CE system to lead large amount sample introduction during EKS. The standard DNA sample of φX174/HaeIII digest was used to evaluate the qualitative and quantitative abilities of the proposed strategy. The 170 000‐fold highly diluted sample at concentration of 3.0 ng/mL was enriched by EKS and detected by normal UV detection method. The obtained LOD of the weakest peak of 72 bp fragment was around 7.7 pg/mL, apparently improved more than 10 000‐fold in comparison with conventional CGE with UV detection.     

Counter-flow electrokinetic supercharging for the determination of non-steroidal anti-inflammatory drugs in water samples

Electrokinetic supercharging (EKS) has been used in the last few years as a powerful tool for separation and on-line preconcentration of different types of analytes. We have developed a valuable modification for EKS system, namely counter-flow EKS (CF-EKS) and applied it for the separation and on-line preconcentration of seven non-steroidal anti-inflammatory drugs (NSAIDs) in water samples. In CF-EKS, a hydrodynamic counter-flow is applied during electrokinetic injection of the analytes within the EKS system. This counter-flow minimises the introduction of the sample matrix into the capillary, allowing longer injections to be performed. Careful choice of the optimum counter-flow as well as the optimum injection voltage allowed the sensitivity to be enhanced by 11,800-fold, giving limits of detection (LODs) of 10.7–47.0 ng/L for the selected NSAIDs. The developed method was validated and then applied for the determination of the studied NSAIDs in drinking water as well as wastewater samples from Hobart city.     

High-sensitivity capillary gel electrophoretic analysis of DNA fragments on an electrophoresis microchip using electrokinetic injection with transient isotachophoretic preconcentration

The research adopted a single-channel microchip as the probe, and focused electrokinetic injection combined with transient isotachophoresis preconcentration technique on capillary electrophoresis microchip to improve the analytical sensitivity of DNA fragments. The channel length, channel width and channel depth of the used microchip were 40.5 mm, and 110 and 50 μm, respectively. The separation was detected by CCD (charge-coupled device) (effective LENGTH=25 mm, 260 nm). A 1/100 diluted sample (0.2 mg/l of each DNA fragment) of commercially available stepladder DNA sample could be baseline separated in 120 s with S/N=2–5. Compared with conventional chip gel electrophoresis, the proposed method is ideally suited to improve the sensitivity of DNA analysis by chip electrophoresis.     

毛细管筛分电泳中电动超荷电结合预填充水塞在线富集十二烷基磺酸钠-蛋白质的复合物

提出了一种应用于毛细管筛分电泳中的电动超荷电结合柱头水塞堆积样品的方法,实现了十二烷基磺酸钠-蛋白质复合物的在线富集。一般情况下,电动超荷电方法是一种将电动进样与瞬时等速电泳联用的富集技术。具体过程是,首先在毛细管中注入背景电解质,再注入适量的前导电解质,然后电动进样一段时间。最后注入后导电解质开始瞬时等速电泳及分离的过程。本文在常规的电动超荷电技术基础上,在电动进样之前先注入一段含有聚合物的水塞以进一步提高富集效果。同时,考察了电动超荷电中不同富集方法叠加联用的效果,包括聚合物的筛分效应、结合水塞和不结合水塞的场放大样品进样效果、瞬时等速电泳等。结果表明,由于十二烷基磺酸钠-蛋白质复合物的质荷比接近,电动进样中的进样歧视得到消除,电动超荷电结合含聚合物水塞堆积样品的方法可以无歧视地在线富集十二烷基磺酸钠-蛋白质复合物,检测灵敏度增强1000倍以上。该方法非常适用于低丰度蛋白质的分析,并可同时提供相对分子质量信息。     

Analysis of phenolic acids by non-aqueous capillary electrophoresis after electrokinetic supercharging

Electrokinetic supercharging (EKS), a new and powerful on-line preconcentration method for capillary electrophoresis, was utilized in non-aqueous capillary electrophoresis (NACE) to enhance the sensitivity of phenolic acids. The buffer acidity and concentration, leader and terminator length and electrokinetic injection time were optimised, with the optimum conditions being: a background electrolyte of 40 mM Tris-acetic acid (pH 7.9), hydrodynamic injection of 50 mM ammonium chloride (22 s, 0.5 psi) as leader, electrokinetic injection of the sample (180 s, -10 kV), hydrodynamic injection of 20 mM CHES (32 s, 0.5 psi) as terminator, before application of the separation voltage (-25 kV). Under these conditions the sensitivity was enhanced between 1333 and 3440 times when compared to a normal hydrodynamic injection with the sample volume <3% of the capillary volume. Detection limits for the seven phenolic acids were in the range of 0.22-0.51 ng/mL and EKS was found to be 3.6-7.9 times more sensitive than large-volume sample stacking and anion selective exhaustive injection for the same seven phenolic acids.     

Electrokinetic supercharging with a system-induced terminator and an optimized capillary versus electrode configuration for parts-per-trillion detection of rare-earth elements in CZE

A further improvement of electrokinetic supercharging (EKS) methodology has been proposed, with the objective to enhance the sensitivity of the conventional CZE-UV method down to a single-digit part per trillion (ppt) level. The advanced EKS procedure is based on a novel phenomenon displaying the formation of a zone with an increased concentration of the hydrogen ion, capable to perform the function of a terminator, behind the sample zone upon electrokinetic injection. In combination with a visualizing co-ion of BGE, protonated 4-methylbenzylamine, acting as the leading ion, such system-induced terminator a effected the transient ITP state to efficiently concentrate cationic analytes prior to CZE. Furthermore, to amass more analyte ions within the effective electric field at the injection stage, a standard sample vial was replaced with an elongated vial that allowed the sample volume to be increased from 500 to 900 μL. Alongside, this replacement made the upright distance between the electrode and the capillary tips prolonged to 40.0 mm to achieve high-efficiency electrokinetic injection. The computer simulation was used for profiling analyte concentration, pH, and field strength in order to delineate formation of the terminator during sample injection. The proposed preconcentration strategy afforded an enrichment factor of 80,000 and thereby the LODs of rare-earth metal ions at the ppt level, e.g. 0.04 nM (6.7 ng/L) for erbium(III).     

Pressure-assisted electrokinetic supercharging for the enhancement of non-steroidal anti-inflammatory drugs

Electrokinetic supercharging (EKS) combines field-amplified sample injection with transient isotachophoresis (tITP) to create a powerful on-line preconcentration technique for capillary electrophoresis. In this work, EKS is enhanced with a positive pressure (pressure-assisted EKS, or PA-EKS) during injection to improve stacking of non-steroidal anti-inflammatory drugs (NSAIDs). Several parameters, including buffer composition and concentration, terminating electrolyte, organic modifier, and injection voltage and injection time of both terminating electrolyte and sample were optimized. Detection limits for seven NSAIDs were determined and an enhancement in sensitivity of almost 50,000-fold was obtained. The PA-EKS method has the potential to be a simple MS compatible preconcentration method to improve the sensitivity of CE.     

Advances of a capillary electrophoretic on-line concentration technique: Electrokinetic supercharging

After comparing with electrokinetic injection (EKI) and transient isotachophoresis (t-ITP), the principles of electrokinetic supercharging (EKS) were introduced. Thereafter, the advances and applications of EKS in capillary electrophoresis were intorduced in the following aspects: EKI setups, t-ITP setups, capillary electrophoresis (CE) separation, and real sample analysis. The factors that limit its application are discussed, and the future development of EKS is also prospected.     

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.     

Highly sensitive and selective separation of intact parathyroid hormone and variants by sheathless CE‐ESI‐MS/MS

Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Sheathless CE‐ESI‐MS, combining CE resolution power and low‐flow ESI sensitivity, was applied to the analysis of PTH in its native conformation in the presence of related forms. Fused silica and neutral‐coated capillaries were investigated, as well as preconcentration methods such as transient isotachophoresis, field‐amplified sample injection (FASI), and electrokinetic supercharging (EKS). The method for the separation of PTH and its variants was first developed using fused‐silica capillary with UV detection. An acidic BGE was used to separate 1–84 PTH (full length), 7–84 PTH, and 1–34 PTH. Acetonitrile was added to the BGE to reduce peptide adsorption onto the capillary wall and transient isotachophoresis was used as analyte preconcentration method. The method was then transferred to a sheathless CE‐ESI‐MS instrument. When using a fused silica capillary, CE‐MS was limited to μg/mL levels. The use of a neutral coating combined with FASI or EKS allowed a significant increase in sensitivity. Under these conditions, 1–84 PTH, 7–84 PTH, and 1–34 PTH were detected at concentrations in the low ng/mL (FASI) or pg/mL (EKS) range.     

Online preconcentration by electrokinetic supercharging for separation of endocrine disrupting chemical and phenolic pollutants in water samples

Online preconcentration using electrokinetic supercharging (EKS) was proposed to enhance the sensitivity of separation for endocrine disrupting chemical (methylparaben (MP)) and phenolic pollutants (2‐nitrophenol (NP) and 4‐chlorophenol (CP)) in water sample. Important EKS and separation conditions such as the concentration of BGE; the choice of terminating electrolyte (TE); and the injection time of leading electrolyte (LE), sample, and TE were optimized. The optimum EKS‐CE conditions were as follows: BGE comprising of 12 mM sodium tetraborate pH 10.1, 100 mM sodium chloride as LE hydrodynamically injected at 50 mbar for 30 s, electrokinetic injection (EKI) of sample at –3 kV for 200 s, and 100 mM CHES as TE hydrodynamically injected at 50 mbar for 40 s. The separation was conducted at negative polarity mode and UV detection at 214 nm. Under these conditions, the sensitivity of analytes was enhanced from 100‐ to 737‐fold as compared to normal CZE with hydrodynamic injection, giving LOD of 4.89, 5.29, and 53 μg/L for MP, NP and CP, respectively. The LODs were adequate for the analysis of NP and CP in environmental water sample having concentration at or lower than their maximum admissible concentration limit (240 and 2000 μg/L for NP and CP). The LOD of MP can be suitable for the analysis of MP exists at mid‐microgram per liter level, even though the LOD was slightly higher than the concentration usually found in water samples (from ng/L to 1 μg/L). The method repeatabilities (%RSD) were in the range of 1.07–2.39% (migration time) and 8.28–14.0% (peak area).     

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.     

Electrokinetic sample injection for high‐sensitivity CZE (part 2): Improving the quantitative repeatability and application of electrokinetic supercharging‐CZE to the detection of atmospheric electrolytes

Electrokinetic supercharging (EKS) is defined as a technique that combines electrokinetic sample injection with transient ITP. Quantitative repeatability of EKS‐CZE and the other CE methods using electrokinetic sample injection process is usually inferior in comparison with the CE methods using hydrodynamic or hydrostatic injection. This is due to some effects, such as the temperature change and the convection of the sample solution in the reservoir, as well as the change of the distance between an electrode and a capillary end (D_ec). In particular, we have found that the D_ec change might most seriously affect the repeatability, especially when the electrode is a thin Pt wire that could be unintentionally bent during sampling. By using a Teflon spacer to fix D_ec to 1.1 mm, the RSD of peak area (n=5) was decreased from 20 to 3.4% in EKS‐CZE for several metal cations. This D_ec dependence of the sample amount injected was supported by computer simulation using CFD‐ACE+ software. The improved repeatability (down to 5.1% at n=5, averaged RSD for Co²⁺, Li⁺, Ni²⁺, Zn²⁺ and Pb²⁺) was also experimentally attained by increasing the D_ec to ca. 20 mm, which was also effective to obtain high sensitivity. Since the temperature and the convection effects on the repeatability are comparatively small in a proper laboratory environment, these effects were estimated from the EKS‐CZE experiments using conditions such as warming and agitating the sample solution during EKS process. Finally, EKS‐CZE was applied to the detection of ions from atmospheric electrolytes in high‐purity water exposed to ambient air for 2 h. The microgram per liter levels of anions (chloride, sulfate, nitrate, formate, acetate and lactate) and cations (ammonium, calcium, sodium and magnesium) could be detected using conventional UV detector.     

Microchip electrophoresis with hydrodynamic injection and waste-removing function for quantitative analysis

Quantitative analysis is problematic for microchip electrophoresis for several reasons including chip-to-chip variation, discontinuous sample re-loading, channel reconditioning, and electrokinetic injection bias. In this study, the capability for quantitative analysis on a flow-through based microchip electrophoresis, which provides continuous sample re-loading, channel washing, reconditioning and hydrodynamic injection as well as waste removing is demonstrated to be more quantifiable and more reproducible compared to manual electrokinetic injection method. Using the flow-through microchip with waste-removing function, FITC-labeled estrogen or Rhodamine B could be continuously analyzed without significant changes (R.S.D. < 6.6%) in signal intensity for over 3 h, which is sufficient for a complete set of quantitative analysis. With the use of a phosphorylated kinase substrate as the model, a calibration curve for quantitative analysis of phosphopeptides were constructed and results indicate that both R2 value of the linearity and R.S.D. values of the peak intensity were around 0.9961 and 3.16%, respectively, without the use of an internal standard. These values were slightly improved to be around 0.9986 and 2.27%, respectively, with the use of a non-phosphopeptide counterpart as the internal standard. The potential of this flow-through device for the development of a kinase phosphorylation assay based on the quantitative method was also briefly discussed.