Native and sodium dodecyl sulfate-capillary gel electrophoresis of proteins on a single microchip

Simultaneous electrophoresis of both native and Sodium dodecyl sulfate (SDS) proteins was observed on a single microchip within 20 min. The capillary array prevented lateral diffusion of SDS components and avoided cross contamination of native protein samples. The planar sputtered electrode format provided a more uniform distribution of separation voltage into each of the 36 parallel microchannel capillaries than platinum wire electrodes commonly used in conventional electrophoresis. The customized geometry of the stacking capillary machined into the cover plate of the microchip facilitated reproducible sample injection without the requirement for stacking gel. Polyimide served as a mask and facilitated insulation of the anode and cathode to prevent electrode lift off and deterioration during continuous electrophoresis, even at a constant current of 8 mA. Improved protein separation was observed during capillary electrophoresis at lower currents. Ferguson plot analysis confirmed the electrophoretic mobility of native globular proteins in accordance with their charge and size. Corresponding Ferguson plot analysis of SDS-associated proteins on the same chip confirmed separation of marker proteins according to their molecular weight.     

Multiplexed DNA sizing by capillary electrophoresis using entangled polymer solutions and diode array detection

We developed a method for the analysis of multiplexed double-stranded DNA (dsDNA) samples complexed to various intercalating dyes using entangled polymer solution. A commercial single-column capillary electrophoresis (CE) instrument with diode array detection was used for multiplexed detection of DNA samples by addition of intercalating fluorescent molecules. A Phi X174HinfI and a pGEM DNA ladder (1 mg/mL) were used for the electrophoretic separation of dsDNA fragments ranging in size from 24 to 726 and 36 to 2645 bp, respectively. The results suggested that simultaneous electrophoretic separation of different DNA ladders multiplexed with different dyes could be performed in the same capillary yielding fast DNA sizing separations. CE analysis, which is often overpowered by slab gel in sample throughput, could now overcome this disadvantage by allowing multiplexed sample analysis in a fraction of the time needed for slab gel analysis. The separation efficiency of stained DNA molecules with both dyes were dramatically improved with buffers containing a large cation such as tetrapentylammonium ion (Npe(4) (+)) as the only cation in the buffer.     

Automated DNA fragment collection by capillary array gel electrophoresis in search of differentially expressed genes

An automatic DNA fragment collector using capillary array gel electrophoresis has been developed. A sheath flow technique is used for not only detection but also collection of DNA fragments. In a sheath flow cell, the DNA fragments separated by 16 capillaries flow independently into corresponding sampling capillaries. The fraction collector consists of 16 sampling trays and each sampling tray is set beneath each end of the sampling capillaries to collect the flow-through DNA fragments. Certain DNA fragments are automatically sorted by controlling the movement of the sampling trays according to the signals from the system. The collector experimentally separated two mixtures of polymerase chain reaction (PCR) products: one prepared by using eight different sizes (base lengths from 161 to 562) of DNAs; and the other prepared by a differential display (DD) method with cDNA fragments. Collected DNA fragments are amplified by PCR and measured by electrophoresis. DNA fragments with base length differences of one (base lengths 363 and 364) were successfully separated. A separated DNA fragment from the DD sample was also successfully sequenced. In addition, differentially expressed DNA fragments were automatically sorted by comparative analysis, in which two similar cDNA fragment groups, labeled by two different fluorophores, respectively, were analyzed in the same gel-filled capillary. These results show that the automatic DNA fragment collector is useful for gene hunting in research fields such as drug discovery and DNA diagnostics.     

Optimizing separation conditions for riboflavin,flavin mononucleotide and flavin adenine dinucleotide in capillary zone electrophoresis with laser-induced fluorescence detection

DNA sequencing is performed in a multiplexed capillary electrophoresis system by UV absorption detection. Four individual electropherograms are obtained by simultaneously running the unlabeled DNA products of the four ddNTP-terminated reactions in the capillary array. The sequence of the template used in the cycle-sequencing reaction can be determined by overlaying the four electropherograms. Two internal standards are employed to adjust for the variance in migration times among the capillaries. After applying the correction algorithm, base calling can be done at a high level of confidence.     

Separating DNA sequencing fragments without a sieving matrix.

The possibility of separating appropriately labeled DNA fragments using free-flow capillary electrophoresis was predicted a few years ago based on simple theoretical arguments. Free-flow separation of double-stranded DNA (dsDNA) fragments in the 100-1000 base range was later demonstrated using a streptavidin label. In this article, we now report that end-labeled free-flow electrophoresis (ELFSE) can also be used to sequence single-stranded DNA (ssDNA). The first 100 bases of a DNA sequencing reaction were read without any sieving matrix when fractionated streptavidin was added to the 5'-end of the ssDNA fragments. These separations required only 18 min and did not require coated capillaries. An analysis of the results indicates that sample injection, analyte-wall interactions and thermal diffusion are the limiting factors at this time. Extrapolating from our data, we predict that several hundred bases could be sequenced in less than 30 min with the proper conditions. ELFSE thus offers an attractive potential alternative to polymer solutions for DNA sequencing in capillaries and microchips.     

Multiplexed detection of nitrate and nitrite for capillary electrophoresis with an automated device for high injection efficiency

A simple automated nanoliter scale injection device which allows for reproducible 5 nL sample injections from samples with a volume of <1 microL is successfully used for conventional capillary electrophoresis (CE) and Hadamard transform (HT) CE detection. Two standard fused silica capillaries are assembled axially through the device to function as an injection and a separation capillary. Sample solution is supplied to the injection capillary using pressure controlled with a solenoid valve. Buffer solution flows gravimetrically by the junction of the injection and separation capillaries and is also gated with a solenoid valve. Plugs of sample are pushed into the space between the injection and separation capillaries for electrokinectic injection. To evaluate the performance of the injection device, several optimizations are performed including the influence of flow rates, the injected sample volume and the control of the buffer transverse flow on the overall sensitivity. The system was then applied to HT-CE-UV detection for the signal-to-noise ratio (S/N) improvement of the nitric oxide (NO) metabolites, nitrite and nitrate. In addition, signal averaging was performed to explore the possibility of greater sensitivity enhancements compared to single injections.     

On-line cation-exchange preconcentration and capillary electrophoresis coupled by tee joint interface

An on-line preconcentration method based on ion exchange solid phase extraction was developed for the determination of cationic analytes in capillary electrophoresis (CE). The preconcentration-separation system consisted of a preconcentration capillary bonded with carboxyl cation-exchange stationary phase, a separation capillary for zone electrophoresis and a tee joint interface of the capillaries. Two capillaries were connected closely inside a 0.3 mm i.d. polytetrafluoroethylene tube with a side opening and fixed together by the interface. The preparations of the preconcentration capillaries and interface were described in detail in this paper. The on-line preconcentration and separation procedure of the analysis system included washing and conditioning the capillaries, loading analytes, filling with buffer solution, eluting analytes and separating by capillary zone electrophoresis (CZE). Several analysis parameters, including sample loading flow rate and time, eluting solution and volume, inner diameter and length of preconcentration capillary etc., were investigated. The proposed method enhanced the detection sensitivity of CE-UV about 5000 times for propranolol and metoprolol compared with normally electrokinetic injection. The detection limits of propranolol and metoprolol were 0.02 and 0.1 microg/L with the proposed method respectively, whereas those were 0.1 and 0.5 mg/L with conventional electrokinetic injection. The experiment results demonstrate that the proposed technique can increase the preconcentration factor evidently.     

Stability of capillary gels for automated sequencing of DNA.

Recent interest in capillary gel electrophoresis has been fueled by the Human Genome Project and other large-scale sequencing projects. Advances in gel polymerization techniques and detector design have enabled sequencing of DNA directly in capillaries. Efforts to exploit this technology have been hampered by problems with the reproducibility and stability of gels. Gel instability manifests itself during electrophoresis as a decrease in the current passing through the capillary under a constant voltage. Upon subsequent microscopic examination, bubbles are often visible at or near the injection (cathodic) end of the capillary gel. Gels have been prepared with the polyacrylamide matrix covalently attached to the silica walls of the capillary. These gels, although more stable, still suffer from problems with bubbles. The use of actual DNA sequencing samples also adversely affects gel stability. We examined the mechanisms underlying these disruptive processes by employing polyacrylamide gel-filled capillaries in which the gel was not attached to the capillary wall. Three sources of gel instability were identified. Bubbles occurring in the absence of sample introduction were attributed to electroosmotic force; replacing the denaturant urea with formamide was shown to reduce the frequency of these bubbles. The slow, steady decline in current through capillary sequencing gels interferes with the ability to detect other gel problems. This phenomenon was shown to be a result of ionic depletion at the gel-liquid interface. The decline was ameliorated by adding denaturant and acrylamide monomers to the buffer reservoirs. Sample-induced problems were shown to be due to the presence of template DNA; elimination of the template allowed sample loading to occur without complications.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-throughput polymerase chain reaction analysis of clinical samples by capillary electrophoresis with UV detection

Routine genetic analysis of large numbers of individuals by polymerase chain reaction (PCR) using capillary electrophoresis is often restricted by the low throughput of standard protocols and the tedious sample preparation process. Here, we demonstrate that capillary electrophoresis with UV detection can be used in PCR-based DNA analysis starting from clinical samples without purification or complicated sample manipulation. After PCR reaction using cheek cells, blood, or HIV-1 gag DNA, the reaction mixtures were injected into a capillary array either on-line or off-line by base stacking. The use of multiplexed absorption detection and the elimination of any purification steps both before and after PCR reaction can potentially provide significant benefits compared to current methods for DNA analysis with regard to time, cost, and labor.     

Capillary array isoelectric focusing with laser-induced fluorescence detection: milli-pH unit resolution and yoctomole mass detection limits in a 32-channel system

We report a multiplexed capillary electrophoresis system employing an array of 32 capillaries with a micromachined sheath-flow cuvette as the detection chamber. The sample streams were simultaneously excited with a 473-nm laser beam, and the fluorescence emission was imaged on a CCD camera with a pair of doublet achromat lens. The instrument produced mass detection limits of 380 ± 120 yoctomoles for fluorescein in zone electrophoresis. Capillary isoelectric focusing of fluorescent standards produced peaks with an average width of 0.0029 ± 0.0008 pH. Capillary coating stability limits the reproducibility of the analysis.     

Capillary electrophoresis of DNA in agarose solutions at 40 degrees C.

DNA fragments ranging from 72 to 1353 bp in length (phi X174 RF DNA/HaeIII) were separated by capillary electrophoresis in 0.3-2.0% solutions of agarose (Sea-Plaque GTG) at 40 degrees C. Liquified agarose above its gelling temperature is easily filled and refilled into capillaries. Its background absorbance at 260 nm was sufficiently low to allow for DNA detection at an estimated DNA load of 13 ng/10 components. Sample injection proceeded at 1 kV for 16 s. The internal capillary diameter was 150 mu, the migration path 27 cm. Migration times varied from 5 to 14 min at 185 V/cm. Potentially, the applicability of capillary electrophoresis in agarose solutions can be expected to extend to the entire size range of DNA, in view of the recent demonstration of kb-sized circular DNA separations in agarose solutions, and those of Mb-sized DNA-agarose complexes in linear polyacrylamide solutions.     

Ultra-slim laminated capillary array for high-speed DNA separation

We developed a new kind of capillary array for electrophoresis by using the numerical-control (NC) wiring technique conventionally used to produce printed-circuit boards. Laminating two polyimide sheets after laying cylindrical capillaries between them according to designed geometries, we fabricated a 16-lane laminated capillary array (LCA) 9.9 cm long, 7.2 cm wide, and 0.5 mm thick in which the effective length of all capillaries was only 10.9 cm. This compact LCA thus had separation columns as short as those in capillary array electrophoresis chips fabricated by lithography techniques. Like conventional capillary arrays, it also enabled pipetting-less direct injection of analytes from sample preparation plates. Using the LCA with LIF detection and a replaceable fluid sieving matrix, we demonstrated high-speed ssDNA fragment separations. At an electric field strength of 316 V/cm, 15 fragments ranging from 50 to 500 bases were completely separated within 5.8 min in all lanes. The lane-to-lane CV of migration time was only 0.38%, and the fragment size for which the resolution per base was 0.59 was 258 +/- 15 bases (average +/-SD).     

Use of disposable open tubular ion exchange pre-columns for in-line clean-up of serum and plasma samples prior to capillary electrophoretic analysis of inorganic cations

A simple analytical system using disposable, open-tubular ion exchange clean-up precolumns coupled in-line to capillary electrophoresis for direct injection of biological samples is presented. The clean-up precolumns were prepared from fused silica capillaries by thermally initiated layer-by-layer polymerization of poly(butadiene-maleic acid) (PBMA) directly on the capillary wall. Typically, 6 cm long precolumns with 4-layers of PBMA were used for sample pretreatment. A robust and reproducible coupling between the precolumn (75 μm ID) and the analytical capillary (50 μm ID) was achieved using an inexpensive, commercially available low dead volume union. No extra dispersion of the analyte zones was observed. Proteins and other high molecular weight compounds from biological sample matrices were retained on the cation-exchanger sites of the precolumn, which eliminated their adsorption on analytical capillary walls and ensured stable electroosmotic flow and migration times of target analytes. Unretained small inorganic cations migrated freely into the analytical capillary for separation and detection. Applicability of the sample clean-up procedure was proved by determination of major inorganic cations in blood serum and plasma samples using capillary electrophoresis with contactless conductivity detection. Separations were performed in background electrolyte solution consisting of 15 mM L-arginine, 12.5 mM maleic acid, 3 mM 18-crown-6 at pH 5.5 and repeatabilities of migration times and peak areas were below 1.5% and 7.3%, respectively. Less than 1 μL of biological sample was required for injection.     

Optimization of affinity capillary electrophoresis for routine investigations of protein–metal ion interactions

To facilitate the implementation of affinity capillary electrophoresis into routine binding screening studies of proteins with metal ions, method acceleration, transfer and precision improvement were investigated. Affinity capillary electrophoresis was accelerated by using shorter capillaries, employing lower sample concentrations and smaller injection volumes. Intra‐ and inter‐instrument method transfers were investigated considering the temperature setting of the capillary cooling system. For intra‐instrument method transfer, similar results were obtained when transferring a method from a long (62 cm) to a short (31 cm) capillary. The analysis time was reduced from 9 to 4 min. In case of inter‐instrument method transfer, interaction results showed small variation on the capillary electrophoresis instrument with inefficient capillary cooling system. Binding measurement precision was enhanced by slightly pushing the sample above the beginning of the capillary. Changing the buffer vials after each 30 runs and employing extra flushing after each 60 subsequent runs further enhanced the precision. The use of 0.1 molar ethylenediaminetetraacetic acid in the rinsing solution successfully desorbs the remaining metal ions from the capillary wall. Excellent precision for apparent mobility ratio measurements was achieved for different protein–metal ion interactions (relative standard deviation of 0.16–0.89%, 15 series, 12 runs for each).     

Influence of the capillary diameter on the separation efficiency and sensitivity: a systematic approach

In this study, the influence of the capillary inner diameter (id) on the efficiency and sensitivity of a capillary zone electrophoresis separation was investigated. Four local anaesthetic drugs (lidocaine, prilocaine hydrochloride, procaine and tetracaine) were separated with a validated method using capillaries of different id. The separation parameter N and the resolution of the critical peak pair were monitored. It was found that N increases in tighter capillaries, while sensitivity decreases indicated by the increased detection limit for lidocaine. This loss in sensitivity can partially be compensated by loading more sample into the capillary by means of an increased injection time and pressure. However, when it comes to the evaluation of the drug quality according to a monograph in the European Pharmacopoeia, we cannot recommend to vary the capillary id in order to meet the system suitability criteria.     

Evaluation of methylation degree from formalin-fixed paraffin-embedded DNA extract by field-amplified sample injection capillary electrophoresis with UV detection

Alterations in global DNA methylation are implicated in several pathobiological processes. The tissues stored as paraffin blocks represent an important source of DNA for retrospective genetic and epigenetic analysis on a large scale. Therefore, we developed the first capillary electrophoresis method able to measure global methylation in formalin-fixed, paraffin-embedded (FFPE) DNA extracts. A field-amplified sample injection capillary electrophoresis method with UV detection for the separation and quantification of cytosine and 5-methylcytosine released following DNA hydrolysis by means of formic acid was employed. Analytes were baseline-separated within 8 min by using 300 mM tris(hydroxymethyl)aminomethane phosphate pH 3.75 as the running buffer. With use of electrokinetic injection the limit of detection (LOD) in real sample was 0.1 nM, thus improving by about 400-fold the LOD of the previously described methods based on capillary electrophoresis. Sample extraction and purification were optimized so that evaluation of the DNA methylation degree was possible starting from 0.5-1 μg of DNA with intra- and interassay relative standard deviations for the 5-methylcytosine to total cytosine ratio of 2.0 and 3.2%, respectively. Because of its high accuracy and throughput, our method will be useful for large-scale applications to determine the implications of genomic DNA methylation levels in tumorigenesis.     

Evaluation of a multiarray system for pharmaceutical analysis by microemulsion electrokinetic chromatography

A multiplexed capillary electrophoresis (CE) system equipped with 96 channels was evaluated for high-throughput screening in drug discovery by microemulsion electrokinetic chromatography (MEEKC). Method transfer from a single channel to a multichannel CE system is described. Loss of efficiency and reduced migration times could be elucidated to the poor efficacy in Joule heat dissipation by forced air cooling in the multiarray system compared to liquid cooling in the single channel instrument. On the other hand, only 48 channels could actually be used because of the maximum total current of 3 mA. Precision data remained below 8% and 9% for migration times and peak areas, respectively. Some UV-detector cross-talk interference between neighboring capillary channels was noted. Impurities at 0.5% compared to the main peak (100%) could be detected with the multiplexed system which is 10 times lower compared to the single capillary system. Higher efficiency and improved figures of merit (absolute sensitivity and no cross-talk interferences) were obtained by using an array of only 24 capillaries.     

Separation and identification of platinum adducts with DNA nucleotides by capillary zone electrophoresis and capillary zone electrophoresis coupled to mass spectrometry

Platinum adducts are supposed to be the cytotoxic lesions in DNA after platinum-containing anticancer therapy. Various adducts are formed upon interaction of platinum complexes with nucleotides, but contribution of individual adducts to antitumor activity and toxicity of platinum complexes still remains to be examined. A capillary zone electrophoresis (CZE) method is described that is suitable to separate individual platinum adducts. We investigated the formation of adducts following the reaction of cis-diamminedichloroplatinum (II) (cisplatin) with various DNA nucleotides. Baseline separation of unmodified and modified nucleotides (adducts) was achieved using uncoated fused-silica capillaries and basic separation buffers. In order to elucidate the observed peak pattern, a coupled CZE-electrospray ionization-mass spectrometry (ESI)-MS approach was applied. After incubation of mononucleotides with cisplatin, monochloro, monoaqua and bifunctional adduct species were detected. Consequently, the migration order of nucleotides and individual platinum adducts could be determined. Moreover, the time-dependent conversion from monochloro to monoaqua and subsequently to bifunctional adducts was monitored. In conclusion, individual platinum adducts were separated by CZE and identified by CZE-ESI-MS. Formation and conversion of distinct species were confirmed. Potential applications comprise studies of novel platinum complexes, investigations of platinum-adduct formation with DNA, and determination of platinum-DNA adducts in cells.     

Micromachined capillary cross-connector for high-precision fraction collection

A new approach for high-precision fraction collection of double-stranded DNA fragments by capillary electrophoresis coupled to a micromachined plastic capillary cross-connector is presented. The system design integrates four fused-silica capillaries with an acrylic cross-channel connector. The cross-channel structure was introduced to enhance the efficiency of the fraction collection process by electrokinetic manipulations. Following the detection of the sample zone of interest at or slightly upstream of the cross during the separation mode, the potentials were reconfigured to collection mode to direct the selected analyte zone into the corresponding collection vial, while keeping the rest of the sample components virtually stopped within the separation capillary. In this way the spacing between consecutive bands of interest can be physically increased, allowing precise isolation of spatially close sample zones. After collection of the target fraction the separation mode is resumed, and the separation/collection cycle is repeated until all desired sample zones are separated and captured. The capillary cross-connector was fabricated of a transparent acrylic substrate by microdrilling flat end and through channels, matching precisely the O.D. and I.D. of the connected capillary tubing, respectively. This design provided a close to zero dead volume connection assembly for the separation and collection capillaries causing minimal extra band broadening during high-precision micropreparative DNA fractionation.     

Capillary zone electrophoresis in laboratory-made fluorinated ethylene propylene capillaries

Capillaries made of fluorinated ethylene propylene (FEP) with an inner diameter of 50 microm have been employed in capillary zone electrophoresis with UV-Vis absorbance detection. The capillaries were made in the laboratory with a recently developed technique using fluoropolymer heat shrink/melt tubing and a tungsten wire as a template for the channel. An electroosmotic flow was obtained in the channels and it is shown that an FEP capillary is more effective for a cationic test solute than a fused-silica capillary. The compatibility of FEP capillaries with optical detection is evaluated briefly.