DNA analysis on microfabricated electrophoretic devices with bubble cells

Microfluidic devices with bubble cells have been fabricated on poly(methyl methacrylate) (PMMA) plates and have been employed for the analysis of DNA using polyethylene oxide (PEO) solutions. First, the separation channel was fabricated using a wire-imprinting method. Then, wires with greater sizes or a razor blade glued in a polycarbonate plate was used to fabricate bubble cells, with sizes of 190-650 microm. The improvements in resolution and sensitivity have been achieved for large DNA (> 603 base pair, bp) using such devices, which depend on the geometry of the bubble cell. The main contributor for optimal resolution is mainly due to DNA migration at lower electric field strengths inside the bubble cell. On the other hand, slight losses of resolution for small DNA fragments have been found mainly due to diffusion, supported by the loss of resolution when separating two small solutes. With a bubble cell of 75 microm (width) x 500 microm (depth), the sensitivity improvement up to 17-fold has been achieved for the 271 bp fragment in the separation of PhiX-174/HaeIII DNA restriction fragments. We have also found that a microfluidic device with a bubble cell of 360 microm x 360 microm is appropriate for DNA analysis. Such a device has been used for separating DNA ranging from 8 to 2176 bp and polymerase chain reaction (PCR) products amplified after 30 cycles, with rapidity and improvements in the sensitivity as well as resolution.     

On-column preconcentration and separation of DNA fragments using polymer solutions in the presence of electroosmotic flow

We demonstrated DNA preconcentration and separation in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions. After injecting large volumes of DNA samples into a capillary filled with free tris(hydroxymethyl)aminomethane (Tris)-borate (TB) buffers, PEO solutions entered the capillary by EOF and acted as sieving matrices. In contrast to conventional methods (in the absence of EOF), controlling the EOF was also useful for resolution optimization. We have found that PEO adsorption on the capillary wall was more pronounced when low ionic strength buffers were used. Thus, the EOF decreased with increasing injection length, which led to longer migration times and changes in resolution and stacking efficiency. All resolution values were higher than 1.5 when 1.0 microg/mL DNA samples were injected at 240 V/cm for 60 s (0.67 microL). In addition, as low as 0.015 microg/mL DNA samples (an about 66-fold increase in sensitivity) were detected when the injection was performed at 250 V/cm for 60 s.     

Comparison of the separation of large DNA fragments in the presence and absence of electroosmotic flow at high pH

This paper describes the analysis of large DNA fragments at pH > 10.0 by capillary electrophoresis (CE) in the presence of electroosmotic flow (EOF) using hydroxyethylcellulose (HEC) solution. HEC solution in the anodic reservoir enters the capillaries filled with high-pH buffer by EOF after sample injection. With respect to resolution, sensitivity, and speed, separation conducted under discontinuous conditions (different pH values of HEC solutions and buffer filling the capillary) is appropriate. Using HEC solution at concentrations higher than its entanglement threshold ensures a good separation of large DNA fragments in the presence of EOF at high pH. In addition to pH and HEC, the electrolyte species, dimethylamine, methylamine, and piperidine, play different roles in determining the resolution. The separation of DNA fragments ranging in size from 5 to 40 kilo base pairs was completed in 6 min using 1.5% HEC prepared in 20 mM methylamine-borate, pH 12.0, and the capillary filled with 40 mM dimethylamine-borate, pH 10.0. In comparison, this method allows faster separations of large DNA fragments compared with that conducted in the absence of EOF using dilute HEC solutions.     

Improving detection in capillary electrophoresis with laser induced fluorescence via a bubble cell capillary and laser power adjustment

Bubble cells have been frequently employed in capillary electrophoresis (CE) to increase the light path length with UV detection to provide an increase in the observed sensitivity of CE; however this approach has not been commonly used for laser-induced fluorescence detection (LIF) with CE. In this paper we study the influence of laser power on the sensitivity of detection in using conventional and enlarged fused silica capillaries for CE with LIF. When using the bubble cell capillary, the laser power must be decreased relative to use of the conventional capillary to reduce the effects of photodegradation of the species being illuminated by the laser. Even though the light intensity was decreased, an increase in sensitivity of detection was observed for most compounds when a bubble cell was used. This increase ranged from a factor of 8 for riboflavin (410 nm excitation) to 3.2 for most aromatic compounds (266 nm excitation), when using a 3x bubble cell compared with a conventional capillary. The bubble cell capillary was used for native detection of IgG by LIF at 266 nm. A limit of detection of 60 ng mL(-1) was obtained from a 20 pg injection, which was 40 times more sensitive than silver staining in conventional SDS/PAGE.     

Analysis of large-volume DNA markers and polymerase chain reaction products by capillary electrophoresis in the presence of electroosmotic flow

We have demonstrated on-line concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions. After injecting large-volumes DNA samples, PEO solutions entered a capillary filled with 400 mM Tris-borate (TB) buffers by EOF and acted as sieving matrices. DNA fragments stacked between the sample zone and PEO solutions. Because sample matrixes affected PEO adsorption on the capillary wall, leading to changes in EOF, migration time, concentration, and resolving power varied with the injection length. When injecting phiX174 RF DNA-HaeIII digest prepared in 5 mM Tris-HCl buffer, pH 7.0, at 250 V/cm, peak height increased linearly as a function of injection volume up to 0.9 microl (injection time 150 s). The sensitivity improvement was 100-fold compare to that injected at 25 V/cm for 10 s (0.006 microl). When injecting 1.54 microl of GeneScan 1000 ROX, the sensitivity improvement was 265-fold. The sensitivity improvement was 40-fold when injecting 0.17 microl DNA sample containing pBR 322/HaeIII, pBR 328/BglI, and pBR 328/HinfI digests prepared in phosphate-buffered saline. This method allows the analysis of polymerase chain reaction (PCR) products amplified after 17 cycles when injecting 0.32 microl (at 30 cm height for 300 s). The total analysis time was shorter (91.6 min) than that (119.6 min) obtained from injecting PCR products after 32 cycles for 10 s.     

Effect of ionic strength, pH and polymer concentration on the separation of DNA fragments in the presence of electroosmotic flow

DNA separations in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions have been demonstrated. During the separations, PEO entered capillaries filled with Tris-borate (TB) free buffers by EOF and acted as sieving matrices. We have found that ionic strength and pH of polymer and free solutions affect the bulk EOF and resolution differently from that in capillary zone electrophoresis. The EOF coefficient increases with increasing ionic strength of the free TB buffers as a result of decreases in the adsorption of PEO molecules. In contrast, the bulk EOF decreases with increasing the ionic strength of polymer solutions using capillaries filled with high concentrations of free TB buffers. Although resolution values are high due to larger differential migration times between any two DNA fragments in a small bulk EOF using 10 mM TB buffers, use of a capillary filled with at least 100 mM TB free buffers is suggested for high-speed separations. On the side of PEO solutions, 1.5% PEO solutions prepared in 100 to 200 mM TB buffers are more proper in terms of resolution and speed. The separation of DNA markers V and VI was accomplished less than 29 min in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 7.0 at 500 V/cm using a capillary filled with 10 mM free TB buffers, pH 7.0.     

Electrophoretic separation of environmentally important phenolic compounds using montomorillonite-coated fused-silica capillaries

This paper reports a simple procedure for coating fused-silica capillaries with poly(diallyldimethyl ammonium chloride) and montmorillonite. The coated capillaries were characterized by performing EOF measurements as a function of buffer pH, number of layers of coating, and number of runs (stability). The coated capillaries showed a highly stable mu(EOF) (run-to-run RSD less than 1.5%, n = 20), allowing continuous use for several days without conditioning. The coated capillaries were then used for the effective separation of nine environmentally important phenolic compounds showing a significant improvement in the resolution, when compared to bare fused-silica capillaries. The EOF of the coated capillaries was constant in alkaline solutions (pH > or = 7), allowing the optimization of the separation conditions of phenolic compounds without significantly affecting the mu(EOF).     

DNA capillary electrophoresis using poly(vinyl alcohol). I. Inner capillary coating

Two new methods of inner capillary coating with poly(vinyl alcohol) (PVAL) have been investigated and evaluated by performing DNA capillary electrophoresis (CE) using PVAL as a separation medium and by measuring the electroosmotic flow (EOF) mobility. The treatment of capillaries with a silanol-group modified PVAL (PVAL-Si) has been found to give good coating effects for improving the resolution of DNA CE and for reducing the EOF. This coating must be effectively achieved by combining the adsorptive property of PVAL chains onto silica with the reaction between the silanol groups of PVAL-Si and the silica surface. The adsorption of PVAL onto silica has been observed by using atomic force microscopy (AFM) for PVAL-Si as well as for a nonmodified PVAL as a control. The coating with PVAL that links to the capillary wall surface with more hydrolytically stable bonding, -Si-C-, has been formed by performing the Grignard reaction, followed by in-capillary polymerization of vinyl acetate (VAc) and hydrolysis. This coating has been found to be effective for improving the resolution of DNA CE and for reducing the EOF.     

On-column concentration and separation of double-stranded DNA by gradient capillary electrophoresis

We describe the separation of dsDNA by capillary electrophoresis in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO). Using 1.0% PEO, the separation of DNA fragments with sizes ranging from 51 bp to 23 kbp has been achieved in less than 12 min, which is better than conventional methods (in the absence of EOF) in terms of speed and resolution. In order to concentrate and separate the DNA sample, gradient changes in the concentrations of PEO and ethidium bromide (EtBr) have been conducted. Different concentrations of PEO solutions are injected to the polyethylene tubes by pressure, where they enter the capillary by EOF. Because the large DNA fragments migrate faster towards the cathode end under counterflow conditions, the introduction sequence is from low to high concentrations of PEO solutions after sample injection. Using the gradient CE approach, the separations of the DNA sample injected at 30 cm height for times up to 120 s have been demonstrated. The linearity between injection time and peak height shows that the DNA fragments stacked during migration from the sample zone to PEO. We found that stacking efficiency is greater when the analysis was performed by simultaneously changing the PEO and EtBr concentration, compared to individual changes in PEO concentration.     

An investigation of peak-broadening effects arising when combining CE with MS

In this study, peak-broadening effects caused by nebulizing gas flow and lack of temperature control have been investigated for separation capillaries with three different inner diameters. The study was performed with serial UV/ESI-MS detection in an effort to distinguish between peak broadening arising in the separation and peak broadening arising in the ion source. The nebulizing gas was found to significantly affect both migration time and separation efficiency when using capillaries with 50 and 75 microm id. If the nebulizing gas is on during injection, the injection volume increases to such an extent that significant peak broadening is induced. Reducing the id to 25 microm minimizes the parabolic flow induced by the nebulizing gas. Results indicate that the nebulizing gas pressure can be optimized to minimize peak broadening in the ion source. A decrease in detection sensitivity, possibly related to the orthogonal design of the interface, was observed when the nebulizing gas pressure was increased. A tapered capillary tip was found to provide superior separation efficiency as well as sensitivity.     

Performance of an in-plane detection cell with integrated waveguides for UV/Vis absorbance measurements on microfluidic separation devices

A microfluidic device with integrated waveguides and a long path length detection cell for UV/Vis absorbance detection is presented. The 750 microm U-cell detection geometry was evaluated in terms of its optical performance as well as its influence on efficiency for electrophoretic separations in the microdevice. Stray light was found to have a strong effect on both, the sensitivity of the detection and the available linear range. The long path length U-cell showed a 9 times higher sensitivity when compared to a conventional capillary electrophoresis (CE) system with a 75 microm inner diameter (ID) capillary, and a 22 times higher sensitivity than with a 50 microm ID capillary. The linear range was comparable to that achieved in a 75 microm ID capillary and more than twice as large as in a 50 microm ID capillary. The use of the 750 microm U-cell did not contribute significantly to band broadening; however, a clear quantification was made difficult by the convolution of several other band broadening sources.     

On-line sample enrichment for the determination of proteins by capillary zone electrophoresis with poly(vinyl alcohol)-coated bubble cell capillaries

Field-amplified sample stacking (FASS) is used to separate basic proteins in a poly-(vinyl alcohol)-coated bubble cell capillary. To our knowledge, this is the first paper describing the on-column stacking of proteins (as cations) using FASS in bubble cell capillary. The bubble cell capillary is fabricated using a one-step method. Cetyltrimethylammonium chloride is added into the running buffer to reverse the EOF and, thus, to pump the water plug out during the sample stacking step. The effect of the water plug lengths and sample injection durations were investigated and optimized. The results obtained were compared with those for the normal capillary without bubble cell in terms of resolution and sensitivity enhancement. Under the optimal condition, this method can improve the sensitivity of the peak areas ranging from 5000- to 26 000-fold. The RSDs (n = 5) of the migration time and peak area are satisfactory (less than 0.6 and 12%, respectively). Application of the capillary electrophoresis method with bubble cell, FASS, and UV detection thereby leads to the determination of these proteins at concentrations ranging from 3 to 10 ng/mL, based on a signal-to-noise ratio of 3:1.     

Interaction between netropsin and double-stranded DNA in capillary zone electrophoresis and affinity capillary electrophoresis

Poor sensitivity and low phase ratio are the main drawbacks of open tubular capillary electrochromatography (OTCEC). The poor sensitivity results from the use of narrow bore size capillary, whereas the low phase ratio, which limits the separation capability, is caused by the limited surface area of conventional capillary. Two strategies may be useful to overcome these disadvantages. First, an extended light path (ELP) capillary, which has a bubble cell at the detection point, is used to improve the sensitivity. Secondly, an etched capillary of a 1,000-fold increased surface area is used to enhance the phase ratio. In this work, use of an ELP capillary and an etched capillary in OTCEC was evaluated with a chiral stationary phase of avidin prepared with the physical adsorption method. With a 20 microm I.D. ELP capillary with a 150 microm bubble cell, the peak height was enhanced by 4-10-fold and the corrected peak area was increased by 12-fold relative to a 20 microm I.D. conventional capillary. However, the peak efficiency and resolution decreased noticeably. The phase ratio on the etched capillary was slightly enhanced, by a factor of 1.64 relative to an unetched capillary. Consequently, the separation capability was slightly improved. The increase in the phase ratio was much lower than that expected from the increase in surface area, the reason for which is probably the reduced density of surface silanol group and the generation of nitrogen-containing groups due to the etching process.     

Lamination-based rapid prototyping of microfluidic devices using flexible thermoplastic substrates

Transposing highly sensitive DNA separation methods (such as DNA sequencing with high read length or the detection of point mutations) to microchip format without loss of resolution requires fabrication of relatively long (approx. 10 cm) microchannels along with sharp injection bands. Conventional soft lithography methods, such as mold casting or hot-embossing in a press, are not convenient for fabricating long channels. We have developed a lamination-based replication technique for rapid fabrication of sealed microfluidic devices with a 10 cm long, linear separation channel. These devices are fabricated in thin cyclo-olefin copolymer (COC) plastic substrates, thus making the device flexible and capable of assuming a range of 3-D configurations. Due to the good optical properties of COC, this new family of devices combines multiple advantages of planar microfluidics and fused-silica capillaries.     

Maximization of injection volumes for DNA analysis in capillary electrophoresis

We report concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solution. DNA fragments migrating against EOF stacked between the sample zone and PEO solution. To maximize the injection volume, several factors, such as concentrations of Tris-borate (TB) buffer and PEO solution, capillary size, and matrix, were carefully evaluated. The use of 25 mM TB buffers, pH 10.0, containing suitable amounts (less than 10 mM) of salts, such as sodium chloride, sodium phosphate, and sodium acetate, to prepare DNA is essential for the concentration of large-volume samples. In the presence of salts, the peaks also became sharper and the fluorescence intensity of DNA complexes increased. Using 2.5% PEO and a 150 microm capillary filled with 400 mM TB buffer, pH 10.0, up to 5 microL DNA samples (phiX 174 RF DNA-HaeIII digest or the mixture of pBR 322/HaeIII, pBR 328/Bg/I, and pBR 328/HinfI digests) have been analyzed, resulting in more than 400-fold improvements in the sensitivity compared to that by conventional injections (ca. 36 nL). Moreover, this method allows the analysis of 3.5 microL PCR products amplified after 17 cycles without any sample pretreatment.     

Performance of throughout in-capillary derivatization capillary electrophoresis employing an on-line sample and run buffer loading device

We report on the effect on performance of varying the length of the capillary during throughout in-capillary derivatization (TICD) capillary electrophoresis (CE). Performance was evaluated by on-line coupling with a sample and CE runbuffer loading device that was newly introduced for this study. The device was assembled with a low cost using two 5 mm inner diameter (ID) disposable polyethylene syringes. First, a sequence was manually formed consisting of a 200 microL run buffer solution plug, a 100 microL sample plug and another 200 microL run buffer solution plug. Each plug was separated from its neighbor by a 100 microL air plug. When each plug reached the injection point where both a platinum-wire anode and the end of the separation capillary tube were located, 340 V/cm separation voltage (electrophoresis voltage) and 34 V/cm injection voltage were applied to the capillary for 3 s. Then the analytes were derivatized during migration in 50 microm ID capillaries filled with 2 mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) in a 20 mM phosphate-borate buffer (pH 10), followed by separating and detecting of OPA derivatives by absorbance of 340 nm. Derivatization, separation, and detection were performed systematically using capillaries which varied in length from 5 to 80 cm. In the case of TICD-CE of a mixture containing 1 mM aspartic acid (Asp) and 20 mM m-nitorophenol (MNP) as a test solution, it was determined that peak area and peak width ratios of Asp to MNP did not depend on capillary length. Enantiomeric separations of DL-alanine (Ala) and Asp were examined using a run buffer consisting of a 45 microM beta-cyclodextrin (CD)-2 mM OPA/NAC-20 mM phosphate-borate buffer (pH 10). Even though the resolution of these enantiomeric pairs decreased with decreasing capillary length, as expected, the peaks corresponding to both enantiomeric amino acids were identified even when a 5 cm capillary was used. An 8-component amino acid mixture was also tested with 5 cm and 10 cm capillaries.     

Novel approach for fritless capillary electrochromatography

At present, the main limitation for the further adoption of capillary electrochromatography (CEC) in the (routine) laboratory is caused by the lack of reproducible and stable columns. The main source of column instability is concentrated in the frits needed to retain the packed bed inside the CEC capillary. The sintering process used to prepare the frits can be rather problematic and irreproducible, particularly for small stationary phase particles and wide column diameters. Since the (surface) composition of the frits is different from the bulk stationary phase packing, different electroosmotic flow (EOF) velocities are generated. This effect is assumed to be primarily responsible for rapid column destruction. In this contribution, a novel approach for the preparation of fritless CEC capillaries is presented and evaluated. Using 5 microm Hypersil ODS particles, separation efficiencies in the range of 130,000-200,000 plates/m were obtained. In a 100 microm inner diameter packed column, electrical currents up to 50 microA could be tolerated without negative effects such as bubble formation. The prepared CEC columns were found to be stable and could easily be operated continuously for several days without column damage. An additional advantage of the proposed tapering approach is that application of pressure on the in- and outlet vial during separation was not required to prevent bubble formation.     

Capillary electrochromatography using continuous-bed columns of sol-gel bonded silica particles with mixed-mode octadecyl and propylsulfonic acid functional groups

Continuous-bed columns containing sol-gel bonded 3 microm silica particles with mixed-mode octadecyl and propylsulfonic acid functional groups (ODS/SCX) were prepared by first packing the ODS/SCX particles into a fused-silica capillary, then filling the packed capillary with a siliceous sol-gel, curing the sol-gel, and finally drying the column with supercritical carbon dioxide. The performance of the sol-gel bonded ODS/SCX columns was evaluated for capillary electrochromatography using acetonitrile aqueous mobile phase containing phosphate buffer. The columns were mechanically strong and permeable. Both EOF velocity and current increased linearly with elevation of the applied electric field strength. The EOF velocity was high at low pH and nearly constant over a range of pH 2-9. Higher buffer concentration resulted in higher current and lower EOF velocity. The acetonitrile content had no significant effect on the EOF. Without thermosetting the column, no bubble formation was noticed with currents up to 2.5 microA. The minimum plate height of a 25/34 cm x 75 microm I.D. sol-gel bonded 3 microm ODS/SCX column was 5.7 microm (1.75 x 10(5) plates per meter) at an optimum EOF velocity of 0.92 mm s(-1). Mixtures of test aromatic compounds and aromatic hydrocarbon homologues gave symmetrical peaks when using a low pH mobile phase. The retention and elution order of aromatic compounds represented a typical reversed-phase separation mechanism similar to conventional ODS columns. The run-to-run and column-to-column retention factor reproducibility was better than 2.5% and 8.0% RSD, respectively.     

A chemometric study of active parameters and their interaction effects in a nebulized sheath-liquid electrospray interface for capillary electrophoresis-mass spectrometry

A chemometrics approach has been used for evaluating the effect of four experimental parameters when coupling capillary electrophoresis (CE) to electrospray ionization-mass spectrometry (ESI-MS). Electrospray voltage, sheath-liquid flow rate, nebulizing gas flow rate, and spray needle position in respect to the MS orifice were varied according to a full factorial design. In addition to main effects, two interaction effects could be identified as significant when measuring the peak intensity of the analytes, from a sample mixture containing peptides and pharmaceuticals. The first interaction effects, between the nebulizing gas flow rate and the sheath-liquid flow rate, and the second interaction effect, between the nebulizing gas flow rate and the spray position, could further explain the impact that these variables have on the spray performance. The number of theoretical plates and the baseline noise were also measured. The sheath-liquid flow was found to significantly affect the separation efficiency, while the noise level mainly was controlled by the nebulizing gas flow. The same factorial design was also used for a CE capillary with lower internal diameter (ID) and the effects of the same variables were compared on those capillaries using equal injection volume for both capillaries. Similar trends were obtained in both capillaries but capillary ID was shown to be a significant variable when evaluating both capillaries in a single model. It was found that a capillary with 25 microm ID provided improved CE-MS performance over than corresponding 50 microm ID capillary. Enhanced sensitivity was obtained using the narrow-bore capillary, and at lower sheath-liquid flow rate the 25 microm ID capillary also gave rise to more efficient peaks.