Electrophoretic analysis of proteins and enantiomers using capillaries modified by a successive multiple ionic-polymer layer (SMIL) coating technique

The applicability of three-layer coatings consisting of three different polymers (A⁺-B⁻-C⁺ coating) prepared by a successive multiple ionic-polymer layer (SMIL) coating technique to the immobilization of polypeptides and/or proteins onto the inner surface of the capillaries was investigated to provide a high-performance separation medium for proteins and enantiomers in capillary electrophoresis (CE). To obtain a stable protein-coated capillary, high molecular mass poly(ethyleneimine) (PEI) was employed as the first layer in the A⁺-B⁻-C⁺ coating, and then a cationic protein was immobilized as the third layer. Comparisons of analytical performances between the A⁺-B⁻-C⁺ coating and the conventional SMIL-coated (A⁺-B⁻-A⁺ coating) capillary were conducted. The CE separation of cationic proteins was successfully achieved with the prepared capillaries. In addition, the polypeptide- and protein-coated capillaries were applied to the chiral separation of a binaphthyl compound. It should be noted that the chiral separation efficiency was strongly dependent on the second anionic polymer layer of the coating. Effects of the interaction between oppositely charged ionic polymer layers on the separation efficiency are discussed.     

Ultraviolet sealing and poly(dimethylacrylamide) modification for poly(dimethylsiloxane)/glass microchips

Simple sealing methods for poly(dimethylsiloxane) (PDMS)/glass-based capillary electrophoresis (CE) microchips by UV irradiation are described. Further, we examined the possibility to modify the inner surface of separation channels, using polymethylacrylamide (PDMA) as a dynamic coating reagent. The surface properties of native PDMS, UV-irradiated PDMS, and PDMA-coated PDMS were systematically studied by atomic force microscopy (AFM), infrared absorption by attenuated total reflection infrared (ATR-IR) spectroscopy, and contact angle measurement. We found that PDMA forms a stable coating on PDMS and glass surfaces, eliminating the nonhomogeneous electroosmotic flow (EOF) in channels on PDMS/glass microchips, and improving the hydrophilicity of PDMS surfaces. Mixtures of flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and fluorescein were separated in 35 s using PDMA-coated PDMS/glass microchips. A high efficiency of theoretical plates with at least 1365 (105 000 N/m) and a good reproducibility with relative standard deviations (RSD) below 4% in five successive separations were achieved.     

Covalent modified hydrophilic polymer brushes onto poly(dimethylsiloxane) microchannel surface for electrophoresis separation of amino acids

A new environmentally friendly method is developed for preventing nonspecific biomolecules from adsorption on poly(dimethylsiloxane) (PDMS) surface via in situ covalent modification. o-[(N-Succinimdyl)succiny]-o'-methyl-poly(ethylene glycol) (NSS-mPEG) was covalently grafted onto PDMS microchannel surface that was pretreated by air-plasma and silanized with 3-aminopropyl-triethoxysilanes (APTES). The modification processes were carried out in aqueous solution without any organic solvent. The mPEG side chains displayed extended structure and created a nonionic hydrophilic polymer brushes layer on PDMS surface, which can effectively prevent the adsorption of biomolecules. The developed method had improved reproducibility of separation and stability of electroosmotic flow (EOF), enhanced hydrophilicity of surface and peak resolution, and decreased adsorption of biomolecules. EOF in the modified microchannel was strongly suppressed, compared with those in the native and silanized PDMS microchips. Seven amino acids have been efficiently separated and successfully detected on the coated PDMS microchip coupled with end-channel amperometric detection. Relative standard deviations (RSDs) of their migration time for run-to-run, day-to-day and chip-to-chip, were all below 2.3%. Moreover, the covalent-modified PDMS channels displayed long-term stability for 4 weeks. This novel coating strategy showed promising application in biomolecules separation.     

Separation of aminophenol isomers in polyelectrolyte multilayers modified PDMS microchip

The separation of three kinds of aminophenol isomers were achieved within 1 min in polyelectrolytes multilayers modified PDMS microchips by layer-by-layer assembly with electrochemical detection (EC). Two polyelectrolytes, poly(dially dimethyl ammonium chloride) (PDDA) and poly(sodium-4-styrene-sulfonate) (PSS) were used to form polyelectrolyte multilayers (PEMs). The surface characteristic of the modified microchip was studied by XPS. The electroosmotic flow (EOF) on PEMs modified PDMS microchips was more stable than that of the native PDMS microchips and the adsorption of samples was greatly reduced on PEMs modified PDMS microchips during the electrophoretic process. The column efficiencies on PEMs modified microchip were increased by 100 times and the signals enhanced by 2 times compared with those of native microchips. The separation conditions such as running buffer pH, running buffer concentration and separation voltage were also optimized.     

Surface modification of poly(dimethylsiloxane) microfluidic devices and its application in simultaneous analysis of uric acid and ascorbic acid in human urine

A novel and simple method based on layer-by-layer (LBL) technique has been developed for the modification of the channel in PDMS electrophoresis microchip to create a hydrophilic surface with a stable EOF. The functional surface was obtained by sequentially immobilizing chitosan and deoxyribonucleic acid (DNA) onto the microfluidic channel surface using the LBL assembly technique. Compared to the native PDMS microchips, the contact angle of the chitosan-DNA modified PDMS microchips decreased and the EOF increased. Experimental conditions were optimized in detail. The chitosan-DNA modified PDMS microchips exhibited good reproducibility and long-term stability. Separation of uric acid (UA) and ascorbic acid (AA) performed on the modified PDMS microchip generated 43,450 and 46,790 N/m theoretical plates compared with 4048 and 19,847 N/m with the native PDMS microchip. In addition, this method has been successfully applied to real human urine samples, without SPE, with recoveries of 97-105% for UA and AA.     

Thermoset polyester as an alternative material for microchip electrophoresis/electrochemistry

Microchip CE coupled with electrochemical detection (MCE-EC) is a good method for the direct detection of many small molecule analytes because the technique is sensitive and readily miniaturized. Polymer materials are being increasingly used with MCE due to their affordability and ease of fabrication. While PDMS has become arguably the most widely used material in MCE-EC due to the simplicity of microelectrode incorporation, it suffers from a lack of separation efficiency, lower surface stability, and a tendency for analyte sorption. Other polymers, such as poly(methylmethacrylate) (PMMA) and poly(carbonate) (PC), have higher separation efficiencies but require more difficult fabrication techniques for electrode incorporation. In this report, thermoset polyester (TPE) was characterized as an alternative material for MCE-EC. TPE microchips were characterized in their native and plasma oxidized forms and after coating with polyelectrolyte multilayers (PEMs). TPE provides higher separation efficiencies when compared to PDMS microchips, while still using simple fabrication protocols. In this work, separation efficiencies as high as 295,000 N/m were seen when using TPE MCE-EC devices. Furthermore, the EOF was higher and more consistent as a function of pH for both native and plasma-treated TPE than PDMS. Finally, TPE is amenable to modification using simple PEM coatings as another way to control surface chemistry and surface charge.     

Hydrophilic biopolymer grafted on poly(dimethylsiloxane) surface for microchip electrophoresis

A novel covalent strategy was developed to modify the poly(dimethylsiloxane) (PDMS) surface. Briefly, dextran was selectively oxidized to aldehyde groups with sodium periodate and subsequently grafted onto amine-functionalized PDMS surface via Schiff base reaction. As expected, the coated PDMS surface efficiently prevented the biomolecules from adsorption. Electro-osmotic flow (EOF) was successfully suppressed compared with that on the native PDMS microchip. Moreover, the stability of EOF was greatly enhanced and the hydrophilicity of PDMS surface was also improved. To apply thus-coated microchip, the separation of peptides, protein and neurotransmitters was investigated in detail. For comparison, these analytes were also measured on the native PDMS microchips. The results demonstrated that these analytes were efficiently separated and detected on the coated PDMS microchips. Furthermore, the relative standard deviations of their migration times for run-to-run, day-to-day, and chip-to-chip reproducibilities were in the range of 0.6-2.7%. In addition, the coated PDMS microchips showed good stability within 1 month.     

Bulk modification of PDMS microchips by an amphiphilic copolymer

A simple and rapid bulk-modification method based on adding an amphiphilic copolymer during the fabrication process was employed to modify PDMS microchips. Poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) was used as the additive substance. Compared to the native PDMS microchips, both the contact angle and the EOF of the bulk-modified PDMS microchips decreased. The effects of the additive loading and the pH on the EOF were investigated in detail. The bulk-modified PDMS microchips exhibited reproducible and stable EOF behavior. The application of the bulk-modified PDMS microchips was also studied and the results indicated that they could be successfully used to separate amino acids and to suppress protein adsorption.     

Comparison of surfactants for dynamic surface modification of poly(dimethylsiloxane) microchips

In the present report, the use of negatively charged surfactants as modifiers of the background electrolyte is reported using poly(dimethylsiloxane) (PDMS) microchips. In particular, the use of anionic surfactants, such as sodium dodecyl sulfate, phosphatidic acid, and deoxycholate, was studied. When surfactants were present in the run buffer, an increase in the electroosmotic flow (EOF) was observed. Two additional effects were also observed: (i) stabilization of the run-to-run EOF, (ii) an improvement in the electrochemical response for several biomolecules. In order to characterize the analysis conditions, the effects of different surfactant, electrolyte, and pH were studied. EOF measurements were performed using either the current monitoring method or by detection of a neutral molecule. The first adsorption/desorption kinetics studies are also reported for different surfactants onto PDMS. The separation of biologically important analytes (glucose, penicillin, phenol, and homovanillic acid) was improved decreasing the analysis time from 200 to 125 s. However, no significant changes in the number of theoretical plates were observed.     

Self-assembled epoxy-modified polymer coating on a poly(dimethylsiloxane) microchip for EOF inhibition and biopolymers separation

A straightforward approach to generate a stable and protein-resistant poly(dimethylsiloxane) (PDMS) surface using self-assembled hydrophilic polymers is demonstrated in this work. Epoxy-modified polymers were directly adsorbed from aqueous solution onto plasma oxidized PDMS based on H-bond interaction, and epoxies of polymer and silanols on oxidized PDMS surface were crosslinked by heating at 110 degrees C. The coating process could be completed within half hour. Poly(dimethylacrylamide-co-glycidyl methacrylate) (PDMA-co-GMA), poly(vinyl pyrrolidone)-g-glycidyl methacrylate (PVP-g-GMA) and poly(vinyl alcohol)-g-glycidyl methacrylate (PVA-g-GMA) (D. P. Wu, B. X. Zhao, Z. P. Dai, J. H. Qin and B. C. Lin, Lab Chip, 2006, 6, 942) were employed as examples here. Unlike PDMA, PVP, and PVA themselves, these epoxy-modified hydrophilic polymers could be directly used as static surface coatings on oxidized PDMS, and inhibited electroosmotic flow (EOF) within pH 3-11. It was also found that hard baking of PDMS at 150 degrees C for 24 hours before surface coating could greatly retard surface hydrophobicity recovery after oxygen plasma exposure, which strengthened epoxy-modified polymer coatings on oxidized PDMS surface, and resulted in EOF less than 0.2 x 10(-4) cm(2) V(-1) s(-1) (pH 9.0) within two weeks. On epoxy-modified polymer coated PDMS microchips, basic proteins, peptides and DNA fragments could be separated satisfactorily, in which more than 2 x 10(4) plates per 2 cm and less than 3% RSD (>8 runs) for migration time were obtained for lysozyme.     

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.     

Study on the separation of amino acids in modified poly(dimethylsiloxane) microchips

A mixture of five amino acids including arginine, histidine, phenylalanine, serine and glutamic acid was successfully separated in microchip capillary electrophoresis and detected with laser-induced fluorescence (LIF) detector. These amino acids were labeled with 5-(4, 6-dichloro-s-triazin-2-ylamino) fluorescein (DTAF). The analyses were performed on two kinds of modified poly(dimethylsiloxane) (PDMS) microchips. One kind of chip was simply treated with oxygen plasma (OP-chip), and the other was further modified by coating double layers of non-ionic polymer poly(vinyl alcohol) (PVA) after plasma oxidization (PVA-chip). The derivatization condition of amino acids by DTAF was optimized. The properties of the two modified PDMS microchips were studied and separation conditions, such as the buffer pH, buffer concentration and separation voltage, were also optimized. The column efficiencies of the two microchips were in the range of 193,000–1,370,000 plates/m. The DTAF-labeled amino acids were sufficiently separated within 50 s and 90 s in 2.5 cm channels on OP-chip and PVA-chip, respectively.     

Use of surface plasmon resonance to study the adsorption of detergents on poly(dimethylsiloxane) surfaces

This paper demonstrates the use of surface plasmon resonance to study adsorption (either reversible or irreversible) of detergents on PDMS surfaces in real time. The surface plasmon resonance measurements can directly provide information about the adsorption/desorption processes of detergents on the surface revealing the durability of the adsorbed layer and the anticipated degree of the EOF. Hydroxypropyl methylcellulose very strongly adsorbs onto PDMS and can be considered both a semipermanent layer and stable semipermanent coating. Adsorbed SDS or CTAB layers were stable for several minutes upon rinsing the surface with solution not containing the detergent. It was shown that SDS coated onto PDMS in microchips has the potential to afford similar separations in PDMS as found in conventional fused silica capillaries.     

Protein separations using polyelectrolyte multilayer coatings with molecular micelles in open tubular capillary electrochromatography

Novel polyelectrolyte multilayer (PEM) coatings for enhanced protein separations in open tubular CEC (OT-CEC) are reported. Use of four cationic polymers (poly-L-lysine, poly-L-ornithine, poly-L-lysine-serine, and poly-L-glutamic acid-lysine), and three anionic molecular micelles, sodium poly(N-undecanoyl-L-leucyl-alaninate) (poly-L-SULA), sodium poly(N-undecanoyl-L-leucyl-valinate) (poly-L-SULV), and sodium poly(undecylenic sulfate) (poly-SUS) were investigated in PEM coatings for protein separations. The simultaneous effects of cationic polymer concentration, number of bilayers, temperature, applied voltage, and pH of the BGE on the separation of four basic proteins (alpha-chymotrypsinogen A, lysozyme, ribonuclease A, and cytochrome c) were analyzed using a Box Behnken experimental design. The influence of NaCl on the run-to-run reproducibility was investigated for PEM coatings containing each cationic polymer. All coatings exhibited excellent reproducibilities with a %RSD of the EOF less than 1% in the presence of NaCl. Optimal conditions were dependent on both the cationic and anionic polymers used in the PEM coatings. Poly-L-glutamic acid-lysine produced the highest resolution and longest migration time. The use of molecular micelles to form PEM coatings resulted in better separations than single cationic coatings. Chiral poly-L-SULA and poly-L-SULV resulted in higher protein resolutions as compared to the achiral, poly-SUS. Furthermore, the use of poly-L-SULV reversed the elution order of lysozyme and cytochrome c when compared to poly-L-SULA and poly-SUS.     

New pressure‐assisted sweeping on‐line preconcentration for highly polar environmentally relevant nitrosamines: Part 2. Cationic and anionic surfactants with zero‐flow capillaries

We recently introduced a pressure‐assisted sweeping‐reversed migration‐EKC (RM‐EKC) method for preconcentration of neutral polar N‐nitrosamines with low affinity for the micellar phase. The type of surfactant and phase ratio are dominant factors in dictating the magnitude of interactions between analyte and micellar phase, thus four surfactants (anionic and cationic) with a range of functionalities (SDS, ammonium perfluorooctanoate (APFO), bile salts, and cetyltrimethylammonium chloride (CTAC)) were evaluated for sweeping‐RM‐EKC of highly polar N‐nitrosamines. All gave acceptable results for sweeping‐RM‐EKC when used in high concentrations (≥200 mM) with low EOF. While no single surfactant was superior by all measures, all but the bile salts had useful performance characteristics. APFO showed the narrowest peak widths and highest number of theoretical plates, though two species co‐migrated at all concentrations (25–300 mM); SDS and the cationic surfactant CTAC also showed good separation characteristics and could resolve all peaks, but CTAC had wider separation window. Various types of capillaries coated for EOF control were compared for use with anionic and cationic surfactants. A commercial zero‐EOF capillary coated with a polymer bearing sulfonic acid functional groups showed superior EOF suppression and reproducibility of migration time with all surfactants.     

Enantiomeric separation by capillary electrophoresis with an electroosmotic flow-controlled capillary

Perfect control of electroosmotic flow (EOF) was achieved by dovetailing successive multiple ionic-polymer layer (SMIL) coated capillaries. The direction and magnitude of the EOF was perfectly controllable over the pH range 2-13. Zone diffusion was not observed, even if the inner wall of the dovetailed capillary was discontinuous, or if the sample zone passed through the connected part of the capillary because the RSDs of migration time, theoretical plates, symmetry factor and S/N of the marker were almost the same when seamless capillary and dovetailed capillary were compared. The dovetailed capillary was applied to cyclodextrin modified capillary zone electrophoresis. The control of the EOF enabled us to control both the resolution and the migration order of the enantiomers. The migration time was also controllable and, therefore, the best condition between separation and migration time could be determined by controlling the EOF. Partial filling affinity electrokinetic chromatography with a protein used as a chiral selector was also studied. The migration of the pseudostationary phase was controllable by EOF, and detection of the solute at 214 nm was possible. Therefore, the EOF-controlled dovetailed capillary has great potential to expand the application of the separation technique.     

On-chip pumping for pressure mobilization of the focused zones following microchip isoelectric focusing

Isoelectric focusing (IEF), traditionally accomplished in slab or tube gels, has also been performed extensively in capillary and, more recently, in microchip formats. IEF separations performed in microchips typically use electroosmotic flow (EOF) or chemical treatment to mobilize the focused zones past the detection point. This report describes the development and optimization of a microchip IEF method in a hybrid PDMS-glass device capable of controlling the mobilization of the focused zones past the detector using on-chip diaphragm pumping. The microchip design consisted of a glass fluid layer (separation channels), a PDMS layer and a glass valve layer (pressure connections and valve seats). Pressure mobilization was achieved on-chip using a diaphragm pump consisting of a series of reversible elastomeric valves, where a central diaphragm valve determined the volume of solution displaced while the gate valves on either side imparted directionality. The pumping rate could be adjusted to control the mobilization flow rate by varying the actuation times and pressure applied to the PDMS to actuate the valves. In order to compare the separation obtained using the chip with that obtained in a capillary, a serpentine channel design was used to match the separation length of the capillary, thereby evaluating the effect of diaphragm pumping itself on the overall separation quality. The optimized mIEF method was applied to the separation of labeled amino acids.     

Interaction of surfactant with antistatic polymer thin layers

Thin layers made from three kinds of hydrophilic polymer were coated onto poly(ethylene terephthalate)(PET) fibers to study the interaction of an anionic surfactant, sodiumn-dodecyl benzenesulfonate, with the polymer layers. The coated layers include a) poly(vinyl alcohol) (PVA) crosslinked with glutaraldehyde [nonionic], b) crosslinked, sulfated PVA [anionic], and c) polyethyleneimine crosslinked with poly(ethyleneglycol diglycidylether) [cationic]. All of these coatings were found to reduce the electrostatic charging of the PET cloths, indicating that they were effectively coated with the hydrophilic polymers. The PET cloth coated with the thin layers was immersed in the aqueous solution of surfactant at 40°C for different durations and the electrostatic voltage as well as the weight change were determined after drying. When the cloth coated with the nonionic or the anionic layer was brought into contact with the surfactant, neither the electrostatic voltage nor the weight of PET changed. On the contrary, immersion in the surfactant solution brought about an increase in both the electrostatic voltage and the weight for the PET coated with the cationic layer. This suggested that the surfactant molecules were bound to the cationic layer, in contrast to the nonionic and the anionic layer. It was concluded that the binding was due to ion complexing between the cationic groups in the polymeric layer and the sulfate groups in the surfactant molecules.     

Investigation of the stability of polyelectrolyte multilayer coatings in open-tubular capillary electrochromatography using laser scanning confocal microscopy

A simple polyelectrolyte multilayer (PEM) coating procedure was used for the development of stable modified capillaries. PEM coatings were constructed in fused-silica capillaries using alternating rinses of cationic and anionic polyelectrolytes. The multilayer coatings investigated in this study consisted of two and twenty layer pairs, or bilayers. A bilayer is one layer of a cationic polymer and one layer of an anionic polymer. Poly(diallyldimethylammonium chloride) was used as the cationic polymer, and the polymeric surfactant poly(sodium N-undecanoyl-L-leucylvalinate) was used as the anionic polymer. Previous studies for both chiral and achiral separations have shown that PEM-coated capillaries have excellent reproducibilities, remarkable endurance, and strong stabilities against extreme pH values when used in open-tubular capillary electrochromatography (OT-CEC). In this study, the stability of the coatings was further investigated after exposure to 0.1 M and 1.0 M NaOH. Structural changes of these coatings were monitored using laser scanning confocal microscopy (LSCM) after flushing the capillaries with NaOH. This technique allowed observation of the degradation of the coatings. Observations are discussed in terms of separations using OT-CEC. Electropherograms obtained from the chiral separation of 1,1'-binaphthyl-2,2'-dihydrogenphosphate in OT-CEC showed a decrease in selectivity and an increase in electroosmotic mobility after long exposure to NaOH. The ability to recover the capillaries by exposure to NaOH was also demonstrated. Measurements of electroosmotic mobility and selectivity showed that 2-bilayer and 20-bilayer PEM coatings could be completely removed from the capillary surface after approximately 3.5 and 9.5 h, respectively, of continuous exposure to 1 M NaOH.     

Electrophoretic separations in poly(dimethylsiloxane) microchips using a mixture of ionic and zwitterionic surfactants

The use of mixtures of ionic and zwitterionic surfactants in poly(dimethylsiloxane) (PDMS) microchips is reported. The effect of surfactant concentration on electroosmotic flow (EOF) was studied for a single anionic surfactant (sodium dodecyl sulfate, SDS), a single zwitterionic surfactant (N-tetradecylammonium-N,N-dimethyl-3-ammonio-1-propanesulfonate, TDAPS), and a mixed SDS/TDAPS surfactant system. SDS increased the EOF as reported previously while TDAPS showed an initial increase in EOF followed by a reduction at higher concentrations. When TDAPS was added to a solution containing SDS, the EOF decreased in a concentration-dependent manner. The EOF for all three surfactant systems followed expected pH trends, with increasing EOF at higher pH. The mixed surfactant system allowed tuning of the EOF across a range of pH and concentration conditions. After establishing the EOF behavior, the adsorption/desorption kinetics were measured and showed a slower adsorption/desorption rate for TDAPS than SDS. Finally, the separation and electrochemical detection of model catecholamines in buffer and reduced glutathione in red blood cell lysate using the mixed surfactant system were explored. The mixed surfactant system provided shorter analysis times and/or improved resolution when compared to the single surfactant systems.