Poly(vinyl alcohol)-coated microfluidic devices for high-performance microchip electrophoresis

The channels of microfluidic glass chips have been coated with poly(vinyl alcohol) (PVA). Applied for microchip electrophoresis, the coated devices exhibited a suppressed electroosmotic flow and improved separation performance. The superior performance of PVA-coated channels could be demonstrated by electrophoretic separations of labeled amines and by video microscopy. While a distorted sample zone is injected using uncoated channels the application of PVA-coated channels results in an improved shape of the sample zone with less band broadening. Applying PVA-coated microchips for the separation of amines labeled with Alexa Fluor 350 even sub-second separations, utilizing a separation length of only 650 microm, could be obtained, while this was not possible using uncoated devices. By using PVA-coated devices rather than an uncoated chip a threefold increase in separation efficiencies could be observed. As the electroosmotic flow (EOF) was suppressed, the anionic compounds were detected at the anode whereas the dominant EOF in uncoated devices resulted in an effective mobility to the cathode. Besides improved separation performance another important feature of the PVA-coated channels was the suppressed adsorption of fluorescent compounds in repetitive runs which results in an improved robustness and detection sensitivity. Applying PVA-coated channels, rinsing or etching steps could be omitted while this was necessary for a reliable operation of uncoated devices.     

Coated microfluidic devices for improved chiral separations in microchip electrophoresis

Chiral separations of fluorescein isothiocyanate-labeled amines have been performed in poly(vinyl alcohol) (PVA)-coated microfluidic glass chips. Baseline separation of enantiomers could be realized in coated devices while they could not be resolved in uncoated chips. The electroosmotic flow (EOF) in PVA-coated channels is suppressed over a wide pH range which leads to a considerable improved reproducibility of migration times in repetitive analysis. Due to the high resolution obtained in such devices, it was possible to reliable determine the enantiomeric purity with high accuracy. One percent of the minor enantiomer could be determined in the presence of large excess of the other enantiomer. As the EOF was suppressed, the anionic compounds were detected at the anode whereas the dominant EOF in uncoated devices resulted in an effective mobility to the cathode. Applying PVA-coated channels considerable improved precision of migration times was found. The relative standard deviation of migration times was below 1% in PVA-coated devices. Accordingly, excessive rinsing or etching steps in order to stabilize the EOF could be omitted while this was necessary for a reliable operation of uncoated devices.     

Separation of somatropin charge variants by multiple‐injection CZE with Polybrene/chondroitin sulfate A double‐coated capillaries

The performance of dynamic double‐coated fused‐silica capillaries with Polybrene and chondroitin sulfate A has been compared with uncoated fused‐silica capillaries for the determination of recombinant human growth factor (somatropin) charge variants. The separations were carried out under the same electrophoretic conditions as described in the European Pharmacopoeia, i.e. at pH 6.0 and 30°C. The coating significantly reduced the interactions between the proteins and the surface of the fused‐silica capillary. The first five separations performed in a new bare fused‐silica capillary were discarded because of very poor separation performance as a result of protein–surface interactions. There was an approximate twofold increase in the interday migration time precision (%RSD ≤ 6.5%) in the double‐coated capillaries. The method was successfully transferred to a multiple CZE mode where two samples were analyzed in a single electrophoretic run. The average purity of somatropin certified reference standard was 98.0% (%RSD ≤ 0.3%) determined by using uncoated and coated capillaries.     

Poly(ethylene glycol)-coated microfluidic devices for chip electrophoresis

Herein, we report on a strategy for durable modification of the channel surface in microfluidic glass chips with the neutral hydrophilic-coating material poly(ethylene glycol) PEG-1M-100. Applied in microchip electrophoresis such PEG-coated devices exhibit a suppressed electroosmotic flow and reduced analyte adsorption. The PEG-coated chips were successfully applied in chip electrophoresis of FITC-labelled amines and amino acids and native proteins as well as in chiral separations. The performance of the coated chips was found to be superior compared with uncoated microchips. The coated chips exhibited high stability and the relative standard deviation of migration times in PEG-coated devices was less than 2%.     

Static adsorptive coating of poly(methyl methacrylate) microfluidic chips for extended usage in DNA separations

A simple and robust static adsorptive (dynamic) coating process using 2% hydroxyethylcellulose was developed for surface modification of poly(methyl methacrylate) (PMMA) microfluidic chips for DNA separations, suitable for usage over extended periods, involving hundreds of runs. The coating medium was also used as a sieving matrix for the DNA separations following the coating process. Four consecutive static treatments, by simply filling the PMMA chip channels with sieving matrix once every day, were required for obtaining a stable coating and optimum performance. The performance of the coated chips at different phases of the coating process was studied by consecutive gel electrophoretic separations with LIF detection using a PhiX-174/HaeIII DNA digest sample. The coated chip, with daily renewal of the sieving matrix, showed high stability in performance during a 25-day period of systematic study, involving more than 100 individual runs. The performance of the coated chip also remained almost the same after 3 months of continuous usage, during which over 200 separations were performed. The average precision of migration time for the 603-bp fragment was 1.31% RSD (n = 6) during the 25-day study, with a separation efficiency of 6.5 x 10(4) plates (effective separation length 5.4 cm).     

Measurement of the differences in electrophoretic mobilities of individual molecules of E. coli beta-galactosidase provides insight into structural differences which underlie enzyme microheterogeneity

The electrophoretic mobility and catalytic activity of individual molecules of Escherichia coli beta-galactosidase were measured using CE-LIF detection. Both the mobility and activity were reproducible for each molecule but differed between individual molecules. Assays were performed using uncoated capillaries and capillaries coated with different polymers, using enzymes from different sources and by three different experimental protocols. In all cases the observed ranges in electrophoretic mobilities were similar. The observed range in the electrophoretic mobility may be explained by structural microheterogeneity resulting in a gain or loss of up to 1.6 suppressed charge units. There was no observed relationship between the observed activities and electrophoretic mobilities. If the finding that individual beta-galactosidase molecules have heterogeneous electrophoretic mobility can be extended to other proteins, this may limit the resolution possible for capillary zone electrophoresis protein separations.     

Surface modification of poly(methyl methacrylate) for improved adsorption of wall coating polymers for microchip electrophoresis

The development of rapid and simple wall coating strategies for high-efficiency electrophoretic separation of DNA is of crucial importance for the successful implementation of miniaturized polymeric DNA analysis systems. In this report, we characterize and compare different methods for the chemical modification of poly(methyl methacrylate) (PMMA) surfaces for the application of wall coating polymers. PMMA surfaces coated with 40 mol% diethylacrylamide and 60 mol% dimethylacrylamide are compared to the PMMA surfaces first oxidized and then coated with hydroxypropylmethyl-cellulose or poly(vinyl alcohol) (PVA). PMMA oxidation was accomplished with UV/ozone or an aqueous solution of HNO(3) to yield hydrogen-bond donors for the spontaneous adsorption of the coating polymers. Contact angle measurements of UV/ozone exposed PMMA surfaces indicate increase in hydrophilicity, and polymer coated surfaces show a strong dependence on the coating polymer and the oxidation method. Fast and repeatable electrophoretic separations of a 10-base and 20-base DNA ladder were performed in PMMA micro CE devices. All analyses were completed in less than 10 min, resulting in the number of theoretical plates as high as 583 000 in a 7.7 cm long separation channel. The duration of UV/ozone treatment was found to have a considerable impact on separation performance. The microchips irradiated with UV for 10 min and coated with PVA as well as the microchips treated with HNO(3) and coated with HPMC were found to have the best separation performance. These results demonstrate facile and robust methods for the surface modification of PMMA enabling low-cost single use devices for electrophoretic DNA separations.     

Investigation of the separability of thaumatin by capillary electrophoresis

The electrophoretic behaviour of the highly basic protein thaumatin was explored in strongly acid (pH 2) and mildly acid (pH 4.5) separation systems using both bare and coated fused silica capillaries. The separation selectivity for thaumatin I, thaumatin II, and for other sample constituents was insufficient for their baseline separation at pH 2 in an uncoated capillary because the separation efficiency was markedly lower than is common in the electrophoretic separations of proteins. A separation selectivity higher by up to one order of magnitude has been reached at pH 4.5. A pronounced asymmetry of zones, which impaired resolution at this pH, was effectively suppressed by coating of the capillary wall with a polymer. In fact, adsorption on the capillary coating always plays a contributory role whenever a good separation of thaumatin constituents is attained. This indicates that electrochromatographic separation systems based on capillaries coated with the layer of either cationic or hydrophilic uncharged polymer hold promise for the development of methods for thaumatin analysis.     

Separation of fluorescein isothiocyanate-labeled amines by microchip electrophoresis in uncoated and polyvinyl alcohol-coated glass chips using water and dimethyl sulfoxide as solvents of background electrolyte

On-chip capillary electrophoresis with uncoated and polyvinyl alcohol-coated glass channels in aqueous and nonaqueous dimethyl sulfoxide (DMSO) background electrolyte (BGE) solutions was applied in the separation of five amines derivatized with fluorescein-5-isothiocyanate. In aqueous BGE at pH 9.2, baseline separation of the analytes was not achieved on uncoated glass chips, but the separation was clearly improved when the chip channels were coated with polyvinyl alcohol (PVA). Separation was successful in nonaqueous DMSO electrolyte solution containing ammonium acetate and sodium methoxide, on both uncoated and PVA-coated glass microchips. The differences in the pK(a) values of analytes were probably amplified in DMSO, and all five analytes were at least partly dissociated and were separated. Because the viscosity of DMSO is higher than that of water, the migration times were longer in DMSO.     

Cross-linked poly(vinyl alcohol) as permanent hydrophilic column coating for capillary electrophoresis.

A fast method for the generation of permanent hydrophilic capillary coatings for capillary electrophoresis (CE) is presented. Such interior coating is effected by treating the surface to be coated with a solution of glutaraldehyde as cross-linking agent followed by a solution of poly(vinyl alcohol) (PVA), which results in an immobilization of the polymer on the capillary surface. Applied for capillary zone electrophoresis (CZE) such capillaries coated with cross-linked PVA exhibit excellent separation performance of adsorptive analytes like basic proteins due to the reduction of analyte-wall interactions. The long-term stability of cross-linked PVA coatings could be proved in very long series of CZE separations. More than 1000 repetitive CE separations of basic proteins were performed with stable absolute migration times relative standard deviation (RSD > 1.2%) and without loss of separation efficiency. Cross-linked PVA coatings exhibit a suppressed electroosmotic flow and excellent stability over a wide pH range.     

Comparison of capillary zone electrophoresis performance of powder-blasted and hydrogen fluoride-etched microchannels in glass

The applicability of glass chips with powder-blasted microchannels for electrophoretic separations was examined, and the performance was compared to microchannels etched with hydrogen fluoride (HF), using bicarbonate buffer and rhodamine B and fluorescein as model compounds. The measured electroosmotic mobilities in all chips were comparable, with values of ca. 7 x 10(-4) cm(2) V(-1)s(-1). The effect of electrical field strength and detection length on the separation efficiency was monitored. It was found that the main source of dispersion is of the Taylor-Aris type, which was discussed in relation to channel roughness differences. Although in powder-blasted channels with a separation length of 8.20 cm, 7-9 times lower plate numbers were obtained than in a HF-etched channel with similar dimensions, successful separation of five fluorescein isothiocyanate (FITC)-labeled amino acids was obtained on a powder-blasted chip within 80 s. Efficiencies of up to 360 000 plates/m were demonstrated on this chip, when a higher buffer concentration was used at a field strength of 664 V/cm. It can be concluded that powder-blasted microchannel chips, although they have a lower separation efficiency compared to HF-etched chips, perform well enough for many applications. Powder blasting can therefore be considered a low-cost and efficient alternative to HF etching, in particular because of the possibility to fabricate access holes through the glass with the same process.     

Micellar electrokinetic chromatography at low pH with polyelectrolyte-coated capillaries

The performance of capillaries coated with a poly(diallyldimethylammonium) (PDADMA) monolayer or poly(diallyldimethylammonium)-poly(styrenesulfonate) bilayer was investigated and compared under micellar electrokinetic chromatographic (MEKC) conditions. Both monolayer (positively charged) and bilayer (negatively charged) coatings with micellar (sodium dodecyl sulfate) electrolyte generated very stable and pH-independent cathodal electroosmotic flow (EOF). From the results obtained, it can be concluded that in a doubly coated capillary the second poly(styrenesulfonate) layer is replaced by sodium dodecyl sulfate micelles during flushing with micellar electrolyte. Consequently, in order to obtain a stable and pH-independent cathodal electroosmotic flow for the MEKC separations, the capillary coating with the second polyanion layer is not necessary. The importance of the PDADMA coating was illustrated by comparing MEKC separations of the common developing agents (hydroquinone, phenidone, pyrocatechol, pyrogallol and quinone) on a bare uncoated capillary with the coated capillary. The coating provides reproducible MEKC separations at low pH (pH 3.0) with relative standard deviation (R.S.D.) values for migration times and peak areas lower than 0.45 and 3.3%, respectively. Good linearities in the range from 5 x 10(-5) to 2 x 10(-3) mol l(-1) were obtained for all five compounds, with correlation coefficients higher than 0.998. The detection limits were in the range from 5 x 10(-6) mol l(-1) for pyrocatechol to 2 x 10(-5) mol l(-1) for quinone. The proposed MEKC system was applied to the determination of hydroquinone and phenidone in X-ray photographic developer solutions.     

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.     

Performance of a novel sieving matrix of poly(vinyl alcohol)/acrylamide copolymer in electrophoretic separations of high molecular weight proteins from red cell membrane

The analysis of high molecular weight (HMW) proteins from complex mixtures is still a challenge in proteomics. This work introduces a novel hydrogel obtained by the copolymerization of an allyl‐PVA derivative with acrylamide and bisacrylamide and applies this matrix to the electrophoretic separation of HMW proteins. By inducing gelation of polyacrylamide in the presence of variable amounts of allyl‐PVA, it is possible to control and vary the average gel porosity. This gel is easy to produce and handle and offers the advantage of being highly mechanically resistant and macroporous. The new matrix was tested in mono‐dimensional separations of complex protein mixtures extracted from red cell membranes with different detergents. The improved performance of this macroporous matrix allowed to identify new proteins by MS and immunoblot analysis using specific antibodies. In particular, the resolution of proteins ranging in size between 97 and 279 kDa was greatly improved here compared to standard polyacrylamide gels, suggesting that this matrix can be a useful tool in routine analysis of HMW proteins in cell biology.     

Free-solution electrophoretic separations of DNA-drag-tag conjugates on glass microchips with no polymer network and no loss of resolution at increased electric field strength

Here, we demonstrate the potential for high-resolution electrophoretic separations of ssDNA-protein conjugates in borosilicate glass microfluidic chips, with no sieving media and excellent repeatability. Using polynucleotides of two different lengths conjugated to moderately cationic protein polymer drag-tags, we measured separation efficiency as a function of applied electric field. In excellent agreement with prior theoretical predictions of Slater et al., resolution is found to remain constant as applied field is increased up to 700 V/cm, the highest field we were able to apply. This remarkable result illustrates the fundamentally different physical limitations of free-solution conjugate electrophoresis (FSCE)-based DNA separations relative to matrix-based DNA electrophoresis. ssDNA separations in "gels" have always shown rapidly declining resolution as the field strength is increased; this is especially true for ssDNA > 400 bases in length. FSCE's ability to decouple DNA peak resolution from applied electric field suggests the future possibility of ultra-rapid FSCE sequencing on chips. We investigated sources of peak broadening for FSCE separations on borosilicate glass microchips, using six different protein polymer drag-tags. For drag-tags with four or more positive charges, electrostatic and adsorptive interactions with poly(N-hydroxyethylacrylamide)-coated microchannel walls led to appreciable band-broadening, while much sharper peaks were seen for bioconjugates with nearly charge-neutral protein drag-tags.     

Improvement of reproducibility and sensitivity of CE analysis by using the capillary coated dynamically with carboxymethyl chitosan

Analysis reproducibility and detection sensitivity of capillary electrophoresis (CE) are often questioned by applied scientists, which has hindered its application as a routine method. To address these issues, a simple, precise, and reproducible dynamic coating method was developed by applying carboxymethyl chitosan (CMC) dynamic coating on fused silica capillary. The proposed coating was accomplished by simply rinsing the capillary with CMC solution for 1 min in between runs, with no regeneration procedure or buffer additives needed. Electroosmotic flow could be well controlled by adjusting the pH of background electrolyte, and the adsorption of analytes onto the capillary inner wall was effectively eliminated. The main parameters of the coating condition were optimized, and extensive applications of these CMC-dynamically coated capillaries in CE separations were then firmly confirmed. By using proteins, aristolochic acids, and inorganic anions as model analytes, the coating showed a good stability, high reproducibility, as well as improved sensitivity. Baseline separations could be obtained with high efficiency. The reduced adsorption was impressively effective for basic proteins, with an average plate number of 90,000/m for each protein, apart from the good resolution on the chromatogram. A high sensitive detection of α-lactalbumin was achieved with a limit of detection (S/N = 3) of 3.5 nM, and the number of theoretical plates was as high as 1,200,000/m. In addition, the combination of the CMC coating with nonaqueous CE and CE-mass spectrometry proved to be practical. All results showed that the CMC-dynamically coated capillary has special properties and obvious superiority over the uncoated ones for CE analysis.     

Enantiomeric analysis of baclofen analogs by capillary zone electrophoresis, using highly sulfated cyclodextrins: Inclusion ionization constant pKa determination

Using cyclodextrin-capillary zone electrophoresis (CD-CZE), baseline separation of baclofen phaclofen, saclofen, and hydroxy-saclofen, potent gamma-aminobutyric acid(B) (GABA(B)) agonist or antagonists was achieved. A method for the enantioresolution of those analogs of GABA was developed using anionic cyclodextrins (highly sulfated CD or highly S-CD) as chiral selectors and capillaries dynamically coated with polyethylene oxide (PEO). With charged CDs we observed good resolutions due to the large electrophoretic mobility of these chiral selectors opposite to the mobility of the solutes. The highly S-alpha-CD and S-beta-CD were found to be complementary and the most effective complexing agent, allowing good enantiomeric resolution in short runtimes. The complete resolution was obtained using 25 mM phosphate buffer at pH 2.5 containing 3% w/v of highly S-alpha-CD or S-beta-CD at 25 degrees C with an applied field of 0.30 kV/cm. The apparent binding constants of the inclusion complexes were evaluated and the migration order was determined. A comparison was possible to investigate the importance of the anionic group of the molecules in the separations. The pK(a) values were determined for all four compounds in order to explain relative electrophoretic migration of the solutes.     

Fast separations with open tubular ion exchange capillary columns

Fused silica columns of 4.6 μm i.d. were coated with 3-sulfopropylsilane as a cation exchanger and 3-(2-aminoethyl-amino)-propylsilane as an anion exchanger. Fast separations of cations (< 25 seconds) and of anions (< 35 seconds) were obtained, using a potentiometric microelectrode as a detector. In proceeding towards smaller i.d.s, a mixture of alkali metal cations could be separated successfully in an uncoated fused silica capillary of 2.3 μm i.d. as a result of retention by the surface silanol groups at pH 9.4.     

Surface modification with BSA blocking based on in situ synthesized gold nanoparticles in poly(dimethylsiloxane) microchip

A stable BSA blocking poly(dimethylsiloxane) (PDMS) microchannel was prepared based on in situ synthesized PDMS–gold nanoparticles composite films. The modified microchip could successfully suppress protein adsorption. The assembly was followed by contact angle, charge-coupled device (CCD) imaging, electroosmotic flow (EOF) measurements and electrophoretic separation methods. Contact angle measurements revealed the coated surface was hydrophilic, water contact angle for coated chips was 45.2° compared to a water contact angle for native PDMS chips of 88.5°. The coated microchips exhibited reproducible and stable EOF behavior. With FITC-labeled myoglobin incubation in the coated channel, no fluorescence was observed with CCD image, and the protein exhibited good electrophoretic effect in the modified microchip.     

Electrophoretic separation of linear and supercoiled DNA in uncoated capillaries.

We report electrophoretic separation of supercoiled plasmids (2-16 kilo base pairs) and linear double-stranded DNA (0.6-23 kilo base pairs) in uncoated capillaries filled with dilute hydroxyethylcellulose. Because electroosmotic flow reverses the order of elution, long plasmids spend less time in the capillary and their bandwidths are narrower than observed in coated capillaries. However, resolution is similar to that obtained in coated capillaries, because it is governed by the distribution of unresolved topoisomers. In the presence of electroosmotic flow migration of supercoiled plasmids does not follow the elastic rod model that has been observed in coated capillaries.