Influence of electric field intensity, ionic strength, and migration distance on the mobility and diffusion in DNA surface electrophoresis

In order to increase the separation rate of surface electrophoresis while preserving the resolution for large DNA chains, e.g., genomic DNA, the mobility and diffusion of Lambda DNA chains adsorbed on flat silicon substrate under an applied electric field, as a function of migration distance, ionic strength, and field intensity, were studied using laser fluorescence microscope. The mobility was found to follow a power law with the field intensity beyond a certain threshold. The detected DNA peak width was shown to be constant with migration distance, slightly smaller with stronger field intensity, but significantly decreased with higher ionic strength. The molecular dynamics simulation demonstrated that the peak width was strongly related with the conformation of DNA chains adsorbed onto surface. The results also implied that there was no diffusion of DNA during migration on surface. Therefore, the Nernst-Einstein relation is not valid in the surface electrophoresis and the separation rate could be improved without losing resolution by decreasing separation distance, increasing buffer concentration, and field intensity. The results indicate the fast separation of genomic DNA chains by surface electrophoresis is possible.     

Quantitative experimental determination of primer-dimer formation risk by free-solution conjugate electrophoresis

DNA barcodes are short, unique ssDNA primers that "mark" individual biomolecules. To gain better understanding of biophysical parameters constraining primer-dimer formation between primers that incorporate barcode sequences, we have developed a capillary electrophoresis method that utilizes drag-tag-DNA conjugates to quantify dimerization risk between primer-barcode pairs. Results obtained with this unique free-solution conjugate electrophoresis approach are useful as quantitatively precise input data to parameterize computation models of dimerization risk. A set of fluorescently labeled, model primer-barcode conjugates were designed with complementary regions of differing lengths to quantify heterodimerization as a function of temperature. Primer-dimer cases comprised two 30-mer primers, one of which was covalently conjugated to a lab-made, chemically synthesized poly-N-methoxyethylglycine drag-tag, which reduced electrophoretic mobility of ssDNA to distinguish it from ds primer-dimers. The drag-tags also provided a shift in mobility for the dsDNA species, which allowed us to quantitate primer-dimer formation. In the experimental studies, pairs of oligonucleotide primer barcodes with fully or partially complementary sequences were annealed, and then separated by free-solution conjugate CE at different temperatures, to assess effects on primer-dimer formation. When less than 30 out of 30 base-pairs were bonded, dimerization was inversely correlated to temperature. Dimerization occurred when more than 15 consecutive base-pairs formed, yet non-consecutive base-pairs did not create stable dimers even when 20 out of 30 possible base-pairs bonded. The use of free-solution electrophoresis in combination with a peptoid drag-tag and different fluorophores enabled precise separation of short DNA fragments to establish a new mobility shift assay for detection of primer-dimer formation.     

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。     

Enzyme enhanced quantitative determination of multiple DNA targets based on capillary electrophoresis

In the current paper, enzyme enhanced simultaneous quantitative determination of multiple DNA targets based on capillary electrophoresis (CE) was described. We used three biotin-modified DNA probes, which reacted with avidin-conjugated horseradish peroxidase (avidin-HRP) conjugate to obtain the HRP labeled probes, to hybridize with three corresponding targets. The resulting mixture containing double-strand DNA (dsDNA)-HRP, excess single-strand DNA (ssDNA)-HRP and remaining avidin-HRP was separated by capillary electrophoresis, and then the system of HRP catalyzing H₂O₂/o-aminophenol (OAP) reaction was adopted. The catalytic product was detected with electrochemical detection. With this protocol, the limits of quantification for the hybridization assay of 21-, 39- and 80-mer DNA fragments were of 1.2 × 10⁻¹¹, 2.4 × 10⁻¹¹ and 3.0 × 10⁻¹¹ M, respectively. The multiplex assay also provided good specificity without any cross-reaction.     

Free-solution electrophoresis of DNA modified with drag-tags at both ends

In end-labeled free-solution electrophoresis (ELFSE), DNA molecules are labeled with a frictional modifier or "drag-tag", allowing their size-based electrophoretic separation in free solution. Among the interesting observations from early work with dsDNA using streptavidin as a drag-tag was that the drag induced by including a streptavidin label at both ends was significantly more than double that from a single streptavidin (Heller, C. et al.., J. Chromatogr. A 1998, 806, 113-121). This finding was assumed to be in error, and subsequent work focused on experiments in which only a single drag-tag is appended to one end of the DNA molecule. Recent theoretical work (McCormick, L. C., Slater, G. W., Electrophoresis 2005, 26, 1659-1667) has examined the contribution of end-effects to the free-solution electrophoretic mobility of charged-uncharged polymer conjugates, reopening the question of enhanced drag from placing a drag-tag at both ends. In this study, this effect is investigated experimentally, using custom-synthesized ssDNA oligonucleotides allowing the attachment of drag-tags to one or both ends, as well as dsDNA PCR products generated with primers appropriate for the attachment of drag-tags at one or both ends. A range of sizes of drag-tags are used, including synthetic polypeptoid drag-tags as well as genetically engineered protein polymer drag-tags. The enhanced drag arising from labeling both ends has been confirmed, with 6-9% additional drag for the ssDNA and 10-23% additional drag for the dsDNA arising from labeling both ends than would be expected from simply doubling the size of the drag-tag at one end. The experimental results for ssDNA labeled at both ends are compared to the predictions of the recent theory of end-effects, with reasonably good quantitative agreement. These experimental findings demonstrate the feasibility of enhancing ELFSE separations by labeling both ends of the DNA molecule, leading to greater resolving power and a wider range of applications for this technique.     

Fast haptoglobin phenotyping based on microchip electrophoresis

A new and fast method for haptoglobin phenotyping was developed based on microchip electrophoresis with laser induced fluorescence detection. Haptoglobin phenotypes 1-1 and 2-2 were labeled with fluorescein isothiocyanate. The analyses were performed on glass microchip which was simply treated with sodium dodecyl sulfate. After the optimization of the separation conditions, Hp 1-1 and Hp 2-2 could be differentiated in 150 s and the detection limits for Hp 1-1 and Hp 2-2 were 0.39 and 0.62 μg/mL, respectively. Finally, the method was applied to human serum samples from healthy people and liver cancer patients. A decrease in Hp concentration for liver cancer patients was confirmed. Featuring high efficiency, speed, simplicity, the method reveals great potentials for the diagnosis of diseases and proteome research.     

Simultaneous detection of 19 K‐ras mutations by free‐solution conjugate electrophoresis of ligase detection reaction products on glass microchips

We demonstrate here the power and flexibility of free‐solution conjugate electrophoresis (FSCE) as a method of separating DNA fragments by electrophoresis with no sieving polymer network. Previous work introduced the coupling of FSCE with ligase detection reaction (LDR) to detect point mutations, even at low abundance compared to the wild‐type DNA. Here, four large drag‐tags are used to achieve free‐solution electrophoretic separation of 19 LDR products ranging in size from 42 to 66 nt that correspond to mutations in the K‐ras oncogene. LDR‐FSCE enabled electrophoretic resolution of these 19 LDR‐FSCE products by CE in 13.5 min (E = 310 V/cm) and by microchip electrophoresis in 140 s (E = 350 V/cm). The power of FSCE is demonstrated in the unique characteristic of free‐solution separations where the separation resolution is constant no matter the electric field strength. By microchip electrophoresis, the electric field was increased to the maximum of the power supply (E = 700 V/cm), and the 19 LDR‐FSCE products were separated in less than 70 s with almost identical resolution to the separation at E = 350 V/cm. These results will aid the goal of screening K‐ras mutations on integrated “sample‐in/answer‐out” devices with amplification, LDR, and detection all on one platform.     

基于毛细管电泳技术的毒品检验与溯源

提出一种基于毛细管电泳技术的毒品检验和溯源方法.针对同一来源和不同来源的没收甲基苯丙胺毒品药片电泳谱图分析,同一批次药品分离得到良好的一致性和重现性(峰高DRS5.3%(n=4);迁移时间DRS1.1%(n=4)),对不同批次的药品的峰高及峰数量有明显区分,实现了通过毛细管电泳达到溯源性分析,为毒品检验及溯源提供了一种可行性方法.     

Electrostatic interaction mechanism on the separation of phenols by non-aqueous capillary electrophoresis

The electrostatic interaction between additive and analyte is of great importance to non-aqueous cap- illary electrophoresis(NACE)separation.Three tetraalkylammonium bromides and acetonitrile were applied as additives and running solvent respectively.The effect of alkyl chain length and concentra- tion of additive on electrostatic interactions was investigated by the separation of phenols.The sepa- ration ability was found to increase with decreasing alkyl chain length of the additive,and the resolu- tion values were increased with increasing additive concentration.The separation was seriously dete- riorated after a little amount of water was added in the running solution.Furthermore,the electrostatic interaction is strong under the conditions of low electron cloud density,weak steric hindrance and multi-interaction sites.Thus,the separation result can be predicted by theoretical analysis,which is helpful for the separation of other substances in NACE based on electrostatic interaction.     

Coupling microchip electrophoresis with MALDI-TOF-MS based on a freezing technique

A freezing technique protocol was proposed for coupling microchip electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALD1-TOF-MS).The microfluidic flow was frozen immediately after electrophoresis on microfluidic chip and the separated analyte molecules were kept in their zone pattern in the electrophoresis.Then,the frozen-chip was lyophilized and sent into TOF-MS instrument as a MALDI target,and the analyte molecules in the microfluidic channels were subjected to analysis by mass spectrometry.This approach could eliminate sample cross-contamination, providing a new interface for microchip electrophoresis and MALDI-MS.     

Study on Rhizoma Chuanxiong based on capillary electrophoresis with amperometric detection

<正>A high-performance capillary electrophoresis with amperometric detection(CE-AD) method has been developed for the analysis of seven bioactive ingredients,namely ferulic acid(FA),vanillin,vanillic acid,p-hydroxybenzoic acid,caffeic acid,gallic acid and protocatechuic acid,in Rhizoma Chuanxiong.The effects of several factors such as the acidity and concentration of running buffer,the separation voltage,the applied potential to working electrode and the injection time were investigated.Under the optimum conditions,the seven analytes could be well separated within 21 min at the separation voltage of 16 kV in a 60 mmol/L borax running buffer(pH 8.7).A 300μm diameter carbon disk electrode has a good response at potential of +950 mV(vs.SCE) for all analytes.Good linear relationship was established over three orders of magnitude with detection limits(S/N = 3) ranged from 3.3×10~(-7) to 6.7×10~(-9)g/mL.This proposed method has been successfully applied for the determination and comparison of different batches of Rhizoma Chuanxiong samples based on their characteristic electrochemical profiles.     

Evaluation of peak overlap in migration-time distributions determined by organelle capillary electrophoresis: Type-II error analogy based on statistical-overlap theory

Organelles commonly are separated by capillary electrophoresis (CE) with laser-induced-fluorescence detection. Usually, it is assumed that peaks observed in the CE originate from single organelles, with negligible occurrence of peak overlap. Under this assumption, migration-time and mobility distributions are obtained by partitioning the CE into different regions and counting the number of observed peaks in each region. In this paper, criteria based on statistical-overlap theory (SOT) are developed to test the assumption of negligible peak overlap and to predict conditions for its validity. For regions of the CE having constant peak density, the numbers of peaks (i.e., intensity profiles of single organelles) and observed peaks (i.e., maxima) are modeled by probability distributions. For minor peak overlap, the distributions partially merge, and their mergence is described by an analogy to the Type-II error of hypothesis testing. Criteria are developed for the amount of peak overlap, at which the number of observed peaks has an 85% or 90% probability of lying within the 95% confidence interval of the number of peaks of single organelles. For this or smaller amounts of peak overlap, the number of observed peaks is a good approximation to the number of peaks. A simple procedure is developed for evaluating peak overlap, requiring determination of only the peak standard deviation, the duration of the region occupied by peaks, and the number of observed peaks in the region. The procedure can be applied independently to each region of the partitioned CE. The procedure is applied to a mitochondrial CE.     

蛋白质的微芯片电泳分离及其与脱氧核糖核酸迁移规律的比较

在自制的聚二甲基硅氧烷(PDMS)微芯片上,使用十二烷基磺酸钠(SDS)无胶筛分电泳分离体系(10 g/L的羟乙基纤维素(HEC), 1 g/L的SDS, 40 mmol/L磷酸盐缓冲溶液,pH 7.0),采用在线自校正激光诱导荧光检测方法,在6.4 min内高效分离了异硫氰酸荧光素(FITC)衍生的6种蛋白质标准样品,连续6次电泳所得迁移时间的相对标准偏差(RSD)均小于10%。用自主建立的脱氧核糖核酸(DNA)定量分离模型对蛋白质迁移数据进行拟合,发现SDS-蛋白质复合物迁移规律与DNA相似,但迁移淌度与相对分子质量及电场强度之间的线性关系明显变差,可见原DNA分离模型要扩展到蛋白质范围必须对一些参数进行校正。     

Comprehensive two-dimensional separations based on capillary high-performance liquid chromatography and microchip electrophoresis

A novel comprehensive two-dimensional (2-D) separation system coupling capillary high-performance liquid chromatography (cHPLC) with microchip electrophoresis (chip CE) is demonstrated. Reversed-phase cHPLC was used as the first dimension, and chip CE acted as the second dimension to perform fast sample transfers and separations. A valve-free gating interface was devised simply by inserting the outlet-end of LC column into the cross-channel on a specially designed chip. A home-made confocal laser-induced fluorescence detector was used to perform on-chip high-sensitive detection. The cHPLC effluents were continuously delivered to the chip and pinched injections of the effluents every 20 seconds were employed for chip CE separation. Gradient elution of cHPLC was carried out to obtain the high-efficiency separation. Free-zone electrophoresis was performed with triethylamine buffer to achieve high-speed separation and prevent sample adsorption. Such a simple-made comprehensive system was proved to be effective. The relative standard deviations for migration time and peak height of rhodamine B in 150 sample transfers were 3.2% and 9.8%, respectively. Peptides of the fluorescein isothiocyanate (FITC)-labeled tryptic digests of bovine serum albumin were fairly resolved and detected with this comprehensive 2-D system.     

A rigid poly(dimethylsiloxane) sandwich electrophoresis microchip based on thin-casting method

We present a novel concept of glass/poly(dimethylsiloxane) (PDMS)/glass sandwich microchip and developed a thin-casting method for fabrication. Unlike the previously reported casting method for fabricating PDMS microchip, several drops of PDMS prepolymer were first added on the silanizing SU-8 master, then another glass plate was placed over the prepolymer as a cover plate, and formed a glass plate/PDMS prepolymer/SU-8 master sandwich mode. In order to form a thin PDMS membrane, a weight was placed on the glass plate. After the whole sandwich mode was cured at 80 degrees C for 30 min, the SU-8 master was easily peeled and the master microstructures were completely transferred to the PDMS membrane which was tightly stuck to the glass plate. The microchip was subsequently assembled by reversible sealing with the glass cover plate. We found that this PDMS sandwich microchip using the thin-casting method could withstand internal pressures of >150 kPa, more than 5 times higher than that of the PDMS hybrid microchip with reversible sealing. In addition, it shows an excellent heat-dissipating property and provides a user-friendly rigid interface just like a glass microchip, which facilitates manipulation of the microchip and fix tubing. As an application, PDMS sandwich microchips were tested in the capillary electrophoresis separation of fluorescein isothiocyanate-labeled amino acids.     

Novel monolithic enzymatic microreactor based on single-enzyme nanoparticles for highly efficient proteolysis and its application in multidimensional liquid chromatography

In this work, a novel and facile monolithic enzymatic microreactor was prepared in the fused-silica capillary via a two-step procedure including surface acryloylation and in situ aqueous polymerization/immobilization to encapsulate a single enzyme, and its application to fast protein digestion through a direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) analysis was demonstrated. At first, vinyl groups on the protein surface were generated by a mild acryloylation with N-acryloxysuccinimide in alkali buffer. Then, acryloylated enzyme was encapsulated into polyacrylates by free-radical copolymerization with acrylamide as the monomer, N,N′-methylenebisacrylamide as the cross-linker, and N,N,N′,N′-tetramethylethylenediamine/ammonium persulfate as the initiator. Finally, polymers were immobilized onto the activated inner wall of capillaries via the reaction of vinyl groups. Capability of the enzyme-immobilized monolithic microreactor was demonstrated by myoglobin and bovine serum albumin as model proteins. The digestion products were characterized using MALDI-TOF-MS with sequence coverage of 94% and 29% observed. This microreactor was also applied to the analysis of fractions through two-dimensional separation of weak anion exchange/reversed-phase liquid chromatography of human liver extract. After a database search, 16 unique peptides corresponding to 3 proteins were identified when two RPLC fractions of human liver extract were digested by the microreactor. This opens a route for its future application in top–down proteomic analysis.     

A novel thermogelling matrix for microchannel DNA sequencing based on poly-N-alkoxyalkylacrylamide copolymers

We have developed a novel class of thermogelling polymer networks based on poly-N-alkoxyalkylacrylamides, and demonstrated their use as DNA sequencing matrices for high-throughput microchannel electrophoresis in capillary arrays. Polymers and copolymers of N-ethoxyethylacrylamide (NEEA) and N-methoxyethylacrylamide (NMEA) were synthesized by aqueous-phase free-radical polymerization and characterized by tandem gel permeation chromatography-multi-angle laser light scattering. These copolymer matrices exhibit "re-entrant"-type volume phase transitions, forming entangled networks with high shear viscosity at low (< 20 degrees C) and high (> 35 degrees C) temperatures, and undergoing a "coil-to-globular", lower critical solution temperature (LCST)-like phase transition over an intermediate temperature range (20-35 degrees C). Hence, matrix viscosity is relatively low at room temperature (25 degrees C), and increases rapidly above 35 degrees C. The material properties and phase behavior of these thermogelling polymer networks were studied by steady-shear rheometry. These matrices are easily loaded into capillary arrays at room temperature while existing as viscous fluids, but thermogel above 35 degrees C to form transparent hydrogels via a thermo-associative phase transition. The extent of the intermediate viscosity drop and the final viscosity increase depends on the composition of the copolymers. DNA sequencing by capillary array electrophoresis with four-color laser-induced fluorescence (LIF) detection shows that these thermogelling networks provide enhanced resolution of both small and large DNA sequencing fragments and longer sequencing read lengths, in comparison to appropriate control (closely related, nonthermogelling) polymer networks. In particular, a copolymer comprised of 90% w/w NMEA and 10% w/w NEEA, with a molecular mass of approximately 2 MDa, delivers around 600 bases at 98.5% base-calling accuracy in 100 min of electrophoresis.     

The molecular end effect and its critical impact on the behavior of charged-uncharged polymer conjugates during free-solution electrophoresis

Recently two novel techniques using free-solution electrophoresis to separate charged-uncharged polymer conjugates have proven successful: end-labeled free-solution electrophoresis (ELFSE) for DNA sequencing, and free-solution conjugate electrophoresis (FSCE) for molar mass profiling of uncharged polymers. The approach taken to analyze the experimental data was an extension of the theory of Long and co-workers (Long, D., Dobrynin, A. V., Rubinstein, M., Ajdari, A., J. Chem. Phys. 1998, 108, 1234-1244) for the electrophoresis of molecules with varying charge distributions. This theory also predicts that the ends of the polymers play a large role in determining the polymer's overall mobility; however, this aspect of the theory was neglected in previous work. Until now this "end effect" has, to the knowledge of the authors, not been recognized in experimental data. Through a careful investigation of the predicted end effect and a reanalysis of the experimental data, we demonstrate that indeed this effect critically impacts on the behavior of charged-uncharged polymer conjugates during electrophoresis. This work indicates that not only does the end effect need to be taken into account to avoid significant errors in data analysis, but also it provides novel system optimization approaches.     

Electrochemical detector based on sol-gel-derived carbon composite material for capillary electrophoresis microchips

An on-chip disk electrode based on sol-gel-derived carbon composite material could be easily and reproducibly fabricated. Unlike other carbon-based electrodes reported previously, this detector is rigid, convenient to fabricate, and amenable to chemical modifications. Based on the stable and reproducible characters of this detector, a copper particle-modified detector was developed for the detection of carbohydrates which extends the application of the carbon-based electrode. In our experiments, the performance of the new integrated detector for rapid on-chip measurement of epinephrine and glucose was illustrated. Experimental procedures including the fabrication of this detector, the configuration of separation channel outlet and electrode verge, and the performance characteristics of this new electrochemical detector were investigated.     

On-line pre-concentration and UV determination of DNA fragments by dynamic coating capillary electrophoresis and its application to detection of genetically modified oilseed rape based on PCR

In this work, it was demonstrated that on-line pre-concentration and separation of DNA fragments within bared silica column by dynamic coating capillary electrophoresis and UV detection. The DNA fragments were pre-concentrated with long electrokinetic injecting time (99 s), peak height increased dramatically as a function of injection time, especially for shorter length DNA. The concentration sensitivity of DNA fragments can be improved from 20- to 100-fold relative to a normal injection (5 s). The electro-osmotic flow (EOF) and DNA-wall interactions within the capillary were eliminated effectively by dynamic coating method. Employing 0.5% poly(ethylene oxide) (PEO) in Tris-phosphate-EDTA (TBE) buffer as sieving matrix, DNA fragments, ranging from 11 to 657 bp, were separated within 20 min. The linear coefficient of linear relation between the migration and DNA length is 0.999. The DNA fragments amplified from transgenic oilseed rape by polymerase chain reaction (PCR) were separated and detected by this method, demonstrating the potential use of this method for effective DNA analysis and detection of genetically modified organisms (GMO).