Ion-permeable membrane for on-chip preconcentration and separation of cancer marker proteins

Cancer marker proteins have been electrophoretically concentrated and then separated in a microfluidic device. On-chip preconcentration was achieved using an ion-permeable membrane, consisting of acrylamide, N,N'-methylene-bisacrylamide and 2-(acrylamido)-2-methylpropanesulfonate. This negatively charged membrane was photopolymerized in the microdevice near the injection intersection. Anionic proteins were excluded from the porous membrane based on both size and charge, which concentrated target components in the injection intersection prior to separation by microchip capillary electrophoresis (μ-CE). Bovine serum albumin was used in the initial characterization of the system and showed a 40-fold enrichment in the μ-CE peak with 4 min of preconcentration. Adjustment of buffer pH enabled baseline resolution of two cancer biomarkers, α-fetoprotein (AFP) and heat shock protein 90 (HSP90), while fine control over preconcentration time limited peak broadening. Our optimized preconcentration and μ-CE approach was applied to AFP and HSP90, where enrichment factors of >10-fold were achieved with just 1 min of preconcentration. Overall, the process was simple and rapid, providing a useful tool for improving detection in microscale systems.     

On-line preconcentration for capillary electrophoresis-atomic fluorescence spectrometric determination of arsenic compounds

An on-line preconcentration method was developed for capillary electrophoresis (CE) with hydride generation-atomic fluorescence spectrometric (HG-AFS) detection of arsenite, arsenate, dimethylarsenic acid, and monomethylarsenic acid. These arsenic species were negatively charged in the sample solution with high pH. When the potential was applied to the electrophoretic capillary, the negatively charged analyte ions moved faster and stacked at the boundary of sample and CE buffer with low pH. So, high sample pH in combination with low buffer pH allowed the injection of large sample volumes (approximately 1100 nL). Comparison of the preconcentration of analyte solution, prepared with doubly deionized water and that prepared with lake or river water, indicated that preconcentration was independent on the original matrix. With injection of approximately 1100 nL sample, an enrichment factor of 37-50-fold was achieved for the four species. Detection limits for the four arsenic species ranged from 5.0 to 9.3 microg.L(-1). Precisions (RSDs, n = 5) were in the range of 4.9-6.7% for migration time, 4.7-11% for peak area, and 4.3-7.1% for peak height, respectively. The recoveries of the four species in locally collected water solution spiked with 0.1 microg.mL(-1) (as As) ranged from 83 to 109%.     

Comparison of sieving matrices for on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments

Commercially available, replaceable sieving matrices and their solvent modulated forms were evaluated for use in on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments in capillary electrophoresis. The fragments were labeled with dyes that can be excited by the 488 nm line of an argon ion laser and have lifetimes in the range of 0.8 ns to 3.8 ns. The sieving matrices and buffer systems included poly(vinylpyrrolidone) (PVP), poly(ethyleneoxide) (PEO), hydroxyethylcellulose (HEC), Tris-borate-EDTA (TBE) and Tris-TAPS-EDTA buffers modified with DMSO and formamide. Selection of the optimal sieving matrix is based on the separation efficiency and the enhancement of lifetime resolution of DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a poly(ethyleneoxide)/TBE gel buffer in the presence of 10% formamide. Received: 25 August 2000 / Revised: 7 November 2000 / Accepted: 14 November 2000     

Matrix conditioning for lengthened capillary DNA sequencing

Capillary electrophoresis (CE) is currently the preferred format for both DNA sequencing and small DNA fragment analysis. The present study provides a simple revision of the procedure used for CE of DNA with a commercial DNA sequencing apparatus from Applied Biosystems. The revision is electrophoretic conditioning of the sieving matrix (typically POP-6) before sample injection. The effects of this preconditioning are revealed during subsequent analyses performed without replenishing the sieving matrix. The primary effect of preconditioning is to increase peak separations during a subsequent CE. The preconditioning has the following characteristics: (i) The effect on peak separation progressively increases as the preconditioning time increases to at least 6 h. (ii) The effect on peak separation scales approximately as the product of the preconditioning time and the magnitude of the electrical field (162 - 320 V/cm) during preconditioning. (iii) The preconditioning persists for more than 72 h at zero field. Preconditioning of the matrix substantially improves resolution of fragment analysis in the range of 700-2000 nucleotides. For DNA sequencing, the primary impact of preconditioning is, thus far, extension of the range of low-quality base calls at the end of sequence reading. Matrix preconditioning is a new factor to consider when interpreting data obtained by CE in polymer solutions. The mechanism of preconditioning is not yet known.     

Mobility-based selective on-line preconcentration of proteins in capillary electrophoresis by controlling electroosmotic flow

A simple method to perform selective on-line preconcentration of protein samples in capillary electrophoresis (CE) is described. The selectivity, based on protein electrophoretic mobility, was achieved by controlling electroosmotic flow (EOF). A short section of dialysis hollow fiber, serving as a porous joint, was connected between two lengths of fused silica capillary. High voltage was applied separately to each capillary, and the EOF in the system was controlled independently of the local electric field intensity by controlling the total voltage drop. An equation relating the EOF with the total voltage drop was derived and evaluated experimentally. On-line preconcentration of both positively charged and negatively charged model proteins was demonstrated without using discontinuous background electrolytes, and protein analytes were concentrated by approximately 60-200-fold under various conditions. For positively charged proteins, positive voltages of the same magnitude were applied at the free ends of the connected capillaries while the porous joint was grounded. This provided a zero EOF in the system and a non-zero local electric field in each capillary to drive the positively charged analytes to the porous joint. CE separation was then initiated by switching the polarity of the high voltage over the second capillary. For negatively charged proteins, the procedure was the same except negative voltages were applied at the free ends of the capillaries. Mobility-based selective on-line preconcentration was also demonstrated with two negatively charged proteins, i.e. beta-lactoglobulin B and myoglobin. In this case, negative voltages of different values were applied at the free ends of the capillaries with different values, which provided a non-zero EOF in the system. The direction of EOF was the same as that of the electrophoretic migration velocities of the protein analytes in the first capillary and opposite in the second capillary. By controlling the EOF, beta-lactoglobulin B, which has a higher mobility, could be concentrated over 150-fold with a 15 min injection while myoglobin, which has a lower mobility, was eliminated from the system.     

Preconcentration and separation of double-stranded DNA fragments by electrophoresis in plastic microfluidic devices

We have evaluated double-stranded DNA separations in microfluidic devices which were designed to couple a sample preconcentration step based on isotachophoresis (ITP) with a zone electrophoretic (ZE) separation step as a method to increase the concentration limit of detection in microfluidic devices. Developed at ACLARA BioSciences, these LabCard trade mark devices are plastic 32 channel chips, designed with a long sample injection channel segment to increase the sample loading. These chips were designed to allow stacking of the sample into a narrow band using discontinuous ITP buffers, and subsequent separation in the ZE mode in sieving polymer solutions. Compared to chip ZE, the sensitivity was increased by 40-fold and we showed baseline resolution of all fragments in the PhiX174/HaeIII DNA digest. The total analysis time was 3 min/sample, or less than 100 min per LabCard device. The resolution for multiplexed PCR samples was the same as obtained in chip ZE. The limit of detection was 9 fg/microL of DNA in 0.1xpolymerase chain reaction (PCR) buffers using confocal fluorescence detection following 488 nm laser excitation with thiazole orange as the fluorescent intercalating dye.     

On-channel base stacking in microchip capillary gel electrophoresis for high-sensitivity DNA fragment analysis

We evaluated a novel strategy for high-sensitivity DNA fragment analysis in a conventional glass double-T microfluidic chip. The microchip allows for a DNA on-channel concentration based on base stacking (BS) with a microchip capillary gel electrophoretic (MCGE) separation step in a poly(vinylpyrrolidone) (PVP) sieving matrix. Depending if low conductivity caused a neutralization reaction between the hydroxide ions and the run buffer component Tris+, the stacking of DNA fragments were processed in the microchip. Compared to a conventional MCGE separation with a normal electrokinetic injection, the peak heights of 50-2650-base pair (bp) DNA fragments on the MCGE-BS separation were increased 3.9-8.0-fold. When we applied the MCGE-BS method to the analysis of a clinical sample of bovine theileria after PCR reaction, the peak height intensity of the amplified 816-bp DNA fragment from the 18S rRNA of T. buffeli was enhanced 7.0-fold compared to that of the normal injection method.     

Enhancement of the conductivity detection signal in capillary electrophoresis systems using neutral cyclodextrins as sweeping agents

Conductivity detection is a universal detection technique often encountered in electrophoretic separation systems, especially in modern chip‐electrophoresis based devices. On the other hand, it is sparsely combined with another contemporary trend of enhancing limits of detection by means of various preconcentration strategies. This can be attributed to the fact that a preconcentration experimental setup usually brings about disturbances in a conductivity baseline. Sweeping with a neutral sweeping agent seems a good candidate for overcoming this problem. A neutral sweeping agent does not hinder the conductivity detection while a charged analyte may preconcentrate on its boundary due to a decrease in its effective mobility. This study investigates such sweeping systems theoretically, by means of computer simulations, and experimentally. A formula is provided for the reliable estimation of the preconcentration factor. Additionally, it is demonstrated that the conductivity signal can significantly benefit from slowing down the analyte and thus the overall signal enhancement can easily overweight amplification caused solely by the sweeping process. The overall enhancement factor can be deduced a priori from the linearized theory of electrophoresis implemented in the PeakMaster freeware. Sweeping by neutral cyclodextrin is demonstrated on an amplification of a conductivity signal of flurbiprofen in a real drug sample. Finally, a possible formation of unexpected system peaks in systems with a neutral sweeping agent is revealed by the computer simulation and confirmed experimentally.     

Electrophoretic chip for high-fidelity fractionation of double-stranded DNA

We report the high fidelity, on-chip fractionation of selected segments from an electrophoretic flow of separated fragments. dsDNA fragments (10-330 base pairs (bp)) were initially separated using a 6.5 cm long channel with an electric field strength of 150 V/cm. As an example of the fractionation process, a target fragment of 20 bp was selected and extracted from the separation channel. The extraction was confirmed and evaluated by fluorescence imaging. High resolution and extraction fidelity were achieved by introducing new procedures for (i) extraction channel-blocking and (ii) segment transfer with cleaning. These procedures are necessary for the development of a practical, fully automated multitarget fractionation electrophoretic chip. A kind of CCD image processing method was introduced to monitor, control, and evaluate the procedure of fractionation. The resolution limits of the separation and extraction are discussed.     

Arsenic speciation by coupling capillary zone electrophoresis with mass spectrometry

Summary Capillary zone electrophoresis (CZE) has been coupled with mass spectrometry to enable the identification of mineral and organometallic compounds of arsenic in speciation studies. The electrophoretic effluent was introduced through a concentric interface into the mass spectrometer. Make-up liquid was added to enable electric contact at the outlet of the separation capillary and to assist the electronebulization process. After ionization, the ions were analyzed and quantified with an ion-trap detector. Optimization of the coupling conditions (geometry of the concentric interface, composition and flow rate of the sheath liquid, electronebulization and detection conditions) is described. The results show that the geometry of the concentric interface and the positioning of the outlet of the separation capillary have a critical effect on stability and sensitivity. Programming the electronebulization and detection conditions throughout the analysis enabled identification and quantification of the seven arsenic compounds of interest (neutral, and positively or negatively charged species) in less than 20 min at the ppm level. Limits of detection ranged from 0.5 to 3.3 mg L⁻¹, corresponding to amounts injected ranging from 15 to 60 pg. The linear dependence of mass spectrometric response on arsenic concentration was verified for concentrations ranging from 5 to 200 mgL⁻¹. For the two positively charged species, arsenobetaine and arsenocholine, an on-line preconcentration technique (field-amplified sample injection) enabled reduction of the detection limits by approximately one order of magnitude to 110 and 160 μgL⁻¹, respectively.     

Characterization of CdSe quantum dots with bidentate ligands by capillary electrophoresis

The CdSe quantum dots (QDs) with bidentate ligands: a-diimine (NN) and dihydrolipoic acid (DHLA) were synthesized and characterized by UV-Vis, particle size and capillary electrophoretic techniques. Two systems were analyzed: CdSe with one ligand (CdSe/ligand) and CdSe with two different ligands (CdSe//ligand1/ligand2), where ligand = α-diimine or DHLA. Hydrodynamic features of functionalized QDs were characterized by zone capillary electrophoretic (CZE), and particle size techniques and these methods were consistent. It was established that CZE, micellar (MEKC) and microemulsion (MEEKC) modes were suitable for separating charged CdSe QDs and that no peaks were obtained for QDs passivated with electrically neutral ligands. For CdSe QDs with neutral (NN) ligands, a preconcentration method with the use of a micellar plug was introduced for visualizing these QDs. A sharp peak representing neutral QDs was obtained within the zone of micellar plug of a non-ionic surfactant, Here, a ligand character used for CdSe modification and the type of the electrophoretic method applied were the determining factors for the QDs peak visualization. Moreover, examples of visualization of charged and neutral QDs on the same run were presented, and for this purpose, dual mechanism (separation/preconcentration) was proposed.      

A novel technique for on-capillary preconcentration of anionic compounds applied to the trace analysis of rapamycin in human blood by capillary electrophoresis

A capillary electrophoretic (CE) method with UV detection at 278 nm has been developed for analysis of the immunosuppressant rapamycin (sirolimus) in human blood at low microg.L(-1) levels. Separation has been achieved in an acidic carrier electrolyte containing sodium dodecyl sulfate and 20% v/v methanol. For sample cleanup and preconcentration, both an off-line solid-phase extraction step using a silica-based reversed-phase material and a newly developed on-capillary focusing technique have been employed. The subsequent treatment of rapamycin under alkaline conditions leads to a cleavage of the lacton bond of the molecule, generating a negatively charged carboxylic group which allows electrokinetic injection into the CE instrument. During the injection process, the negatively charged analyte migrates into an acidic carrier electrolyte, so that it becomes neutral due to protonation and is focused at the capillary inlet. Injection times of 300 s at -7.5 kV could be applied without band-broadening. Results for real samples indicated that the method is fully suited for routine applications and may be an attractive alternative to established liquid chromatographic techniques.     

Strategies for the on-line preconcentration and separation of hypolipidaemic drugs using micellar electrokinetic chromatography

Three strategies were investigated for the simultaneous separation and on-line preconcentration of charged and neutral hypolipidaemic drugs in micellar electrokinetic chromatography (MEKC). A background electrolyte (BGE) consisting of 20 mM ammonium bicarbonate buffer (pH 8.50) and 50 mM sodium dodecyl sulfate (SDS) was used for the separation and on-line preconcentration of the drugs. The efficiencies of sweeping, analyte focusing by micelle collapse (AFMC), and simultaneous field-amplified sample stacking (FASS) and sweeping, were compared for the preconcentration of eight hypolipidaemic drugs in different conductivity sample matrices. When compared with a hydrodynamic injection (5 s at 50 mbar, 0.51% of capillary volume to detection window) of drug mixture prepared in the separation BGE, improvements of detection sensitivity of 60-, 83-, and 80-fold were obtained with sweeping, AFMC and simultaneous FASS and sweeping, respectively, giving limits of detection (LODs) of 50, 36, and 38 μg/L, respectively. The studied techniques showed suitability for focusing different types of analytes having different values of retention factor (k). This is the first report for the separation of different types of hypolipidaemic drugs by capillary electrophoresis (CE). The three methods were validated then applied for the analysis of target analytes in wastewater samples from Hobart city.     

压力对加压电色谱分离选择性影响的研究

基于弛豫理论的基本原理,通过对溶质在加压电色谱中输运过程的研究,得到了溶质多种形态同时存在情况下反映其色谱行为与电泳行为对迁移时间影响的理论表达式。进一步研究了中性溶质与中性溶质、中性溶质与带电溶质以及带电溶质与带电溶质的分离选择性与压力、操作电压等的关系,结果表明：对于中性溶质的分离,尽管可以通过压力的改变调节峰间距和分离时间,但不会对出峰次序产生影响。对于中性溶质与带电溶质的分离以及带电溶质与带电溶质的分离,压力和电压皆可以便利地用于选择性的调节。     

Electrokinetic molecular separation in nanoscale fluidic channels

This report presents a study of electrokinetic transport in a series of integrated macro- to nano-fluidic chips that allow for controlled injection of molecular mixtures into high-density arrays of nanochannels. The high-aspect-ratio nanochannels were fabricated on a Si wafer using interferometric lithography and standard semiconductor industry processes, and are capped with a transparent Pyrex cover slip to allow for experimental observations. Confocal laser scanning microscopy was used to examine the electrokinetic transport of a negatively charged dye (Alexa 488) and a neutral dye (rhodamine B) within nanochannels that varied in width from 35 to 200 nm with electric field strengths equal to or below 2000 V m-1. In the negatively charged channels, nanoconfinement and interactions between the respective solutes and channel walls give rise to higher electroosmotic velocities for the negatively charged dye than for the neutral dye, towards the negative electrode, resulting in an anomalous separation that occurs over a relatively short distance (<1 mm). Increasing the channel widths leads to a switch in the electroosmotic transport behavior observed in microscale channels, where neutral molecules move faster because the negatively charged molecules are slowed by the electrophoretic drag. Thus a clear distinction between "nano-" and "microfluidic" regimes is established. We present an analytical model that accounts for the electrokinetic transport and adsorption (of the neutral dye) at the channel walls, and is in good agreement with the experimental data. The observed effects have potential for use in new nano-separation technologies.     

Comparison of sieving matrices for on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments

Commercially available, replaceable sieving matrices and their solvent modulated forms were evaluated for use in on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments in capillary electrophoresis. The fragments were labeled with dyes that can be excited by the 488 nm line of an argon ion laser and have lifetimes in the range of 0.8 ns to 3.8 ns. The sieving matrices and buffer systems included poly(vinylpyrrolidone) (PVP), poly(ethyleneoxide) (PEO), hydroxyethylcellulose (HEC), Tris-borate-EDTA (TBE) and Tris-TAPS-EDTA buffers modified with DMSO and formamide. Selection of the optimal sieving matrix is based on the separation efficiency and the enhancement of lifetime resolution of DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a poly(ethyleneoxide)/TBE gel buffer in the presence of 10% formamide.     

Separation of negatively charged nonsteroidal anti-inflammatory drugs by reversed-phase capillary electrochromatography

Reversed-phase capillary electrochromatography in a 5-microm C18 fully packed capillary was employed to optimize the separation of negatively charged nonsteroidal anti-inflammatory drugs. The effect of the physico-chemical parameters and different analysis modes on the separation of 2-arylpropionic acids was studied and evaluated. The mobile phase composition, buffer type, concentration and pH differently influenced the peak efficiency and resolution, selectively modulating the analytes interaction with the stationary phase. The use of zwitterionic MES or acetate mobile phases strongly modulated the analytes migration order and peak efficiency. The optimum experimental conditions were found in MES buffer, pH 5.0, containing the 75% acetonitrile-methanol (1:1). All the analytes were baseline separated in a mixture in less than 13 min with peak efficiencies in the range of 78,500-84,200 N/m. Under these conditions the analytes were negatively charged and their effective electrophoretic mobilities played a role in the separation. The analysis of different pharmaceutical preparations containing anti-inflammatory drugs, e.g. drops and tablets, is also presented after a very simple sample pretreatment.     

Temperature-dependence of preconditioning for lengthened capillary DNA sequencing

A previous study shows that electrophoretic preconditioning of a commercial polymer solution increases the spacing and resolution of DNA fragments fractionated in this solution by CE at 50 degrees C (Griess, G. A. et al., Electrophoresis 2005, 26, 102). The present study shows that this preconditioning effect on peak spacing progressively increases when the temperature of preconditioning increases to 70 degrees C, though fractionation is still performed at 50 degrees C. An increase in peak sharpness accompanies the increase in peak separation for DNA fragments longer than 200 bases. Changing the preconditioning temperature from 50 to 70 degrees C optimally improves resolution of fragment analysis in the range of 600-2000 nucleotides. When DNA sequencing is performed with automated base calling and 70 degrees C preconditioning at 319 V/cm (47 cm long capillary, Applied Biosystems 310 apparatus), the range of high-quality base calls is increased by 25% to 750; the range of low-quality base calls is increased by about 100% to 1200 in comparison to DNA sequencing without preconditioning.     

On-line preconcentration of organic anions in capillary electrophoresis by solid-phase extraction using latex-coated monolithic stationary phases

Quaternary ammonium functionalised polymeric latex particles were coated onto the wall of a fused-silica capillary or onto a methacrylate monolithic bed synthesised inside the capillary in order to create ion-exchange stationary phases of varying ion-exchange capacity. These capillaries were coupled in-line to a separation capillary and used for the solid-phase extraction (SPE), preconcentration and subsequent separation of organic anions by capillary electrophoresis. A transient isotachophoretic gradient was used for the elution of bound analytes from the SPE phase using two modes of separation. The first comprised a low capacity SPE column combined with a fluoride/octanesulfonate discontinuous electrolyte system in which peak compression occurred at the isotachophoretic gradient front. The compressed anions were separated electrophoretically after elution from the SPE preconcentration phase and resolution was achieved by altering the pH of the electrolyte in which the separation was performed. In the second approach, a latex-coated monolithic SPE preconcentration stationary phase was used in combination with a fluoride/perchlorate electrolyte system, which allowed capillary electrochromatographic separation to occur behind the isotachophoretic gradient front. This method permitted the removal of weakly bound anions from the SPE phase, thereby establishing the possibility of sample clean-up. The effect of the nature of the strong electrolyte forming the isotachophoretic gradient on the separation and also on the preconcentration step was investigated. Capillary electrochromatography of inorganic and organic species performed on the latex-coated monolithic methacrylate column highlighted the presence of mixed-mode interactions resulting from the incomplete coverage of latex particles onto the monolithic surface. Analyte preconcentration prior to separation resulted in compression of the analyte zone by a factor of 300. Improvement in the limit of detection of up to 10400 times could be achieved when performing the preconcentration step and the presented methods had limits of detection (S/N=3) ranging between 1.5 and 12 nM for the organic anions studied.     

Enantioseparation of dihydropyridine derivatives by means of neutral and negatively charged beta-cyclodextrin derivatives using capillary electrophoresis

Employing capillary electrophoresis, the racemates of 29 acidic, neutral and basic dihydropyridines (DHPs) were separated by means of neutral and negatively charged cyclodextrins (CDs). Whereas the enantiomers of the acidic DHPs could be resolved with neutral CDs, mostly alpha- and beta-CD, the enantiomers of the neutral DHPs were only baseline-separated using the sulfobutyl ether-substituted beta-CD. Working in reversed polarity mode (detector at the anode) improved the peak shape and the resolution of the enantiomers. The racemates of the DHP bearing a secondary or tertiary amine function in the side chain at position 3 could be separated by using either the neutral gamma-CD or negatively charged CDs. The poor peak shape found with anionic CDs could be improved by the addition of methanol. The combination of gamma-CD and sulfated beta-CD allowed the detection of the minor enantiomer of lercanidipine (24) at less than 1% w/w.