Electrokinetic supercharging preconcentration and microchip gel electrophoretic separation of sodium dodecyl sulfate-protein complexes

A microchip gel electrophoresis (MCGE) method with electrokinetic supercharging (EKS, electrokinetic injection with transient isotachophoresis) on a single channel chip was developed for high-sensitive detection of a standard mixture of six proteins (phosphorylase b, albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor, and alpha-lactalbumin) in the form of sodium dodecyl sulfate (SDS) complexes. An average lower limit of detectable concentration (LLDC) achieved using UV detection at 214 nm was 0.27 microg/mL that is 30 times lower than that of conventional MCGE on a cross geometry chip. The calibration curves for molecular weight and concentration of SDS-protein complexes suggested that the present EKS-MCGE method had a better linear dynamic range and benefited future applications for qualitative and quantitative analysis of unknown protein samples. It was found that an excessive amount of unbound SDS in the sample deteriorated the preconcentration effect and resolution. The developed method appears greatly promising for high-speed and high sensitive analysis of SDS-proteins by MCGE.     

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.     

Online transient isotachophoresis concentration by the pseudo-terminating electrolyte buffer for the separation of DNA-aptamer and its thrombin complex in poly(methyl methacrylate) microchip

Online automatic transient isotachophoresis concentration of DNA-aptamer and its thrombin complex by using one kind of pseudo-terminating electrolyte buffer in a cross-channel poly(methyl methacrylate) microchip is reported. Sample injection, transient concentration and separation were done continuously and controlled by a sequential voltage switching program, time-consuming steps and complicated chip design were not required. Peak resolution between DNA-aptamer and its thrombin complex was influenced by this novel pseudo-terminating electrolyte buffer, which was prepared by the addition of chemical component with slow mobility into the same buffer as leading electrolyte buffer. 1100-fold signal enhancement of thrombin complex was achieved by this transient isotachophoresis on a standard cross-form microchip. The concentration effect or standing time of transient isotachophoresis was proved to be influenced by the concentration of leading electrolyte ion and the concentration of pseudo-terminating electrolyte buffer ion (glycine). The transient concentration was followed by on-chip nondenaturing gel electrophoresis in methylcellulose solution for the size-based separation. The detection limit, taken as the lowest thrombin concentration at threefold S/N, was determined to be 0.5 amol in mass by this method.     

A sol-gel-modified poly(methyl methacrylate) electrophoresis microchip with a hydrophilic channel wall

A sol-gel method was employed to fabricate a poly(methyl methacrylate) (PMMA) electrophoresis microchip that contains a hydrophilic channel wall. To fabricate such a device, tetraethoxysilane (TEOS) was injected into the PMMA channel and was allowed to diffuse into the surface layer for 24 h. After removing the excess TEOS, the channel was filled with an acidic solution for 3 h. Subsequently, the channel was flushed with water and was pretreated in an oven to obtain a sol-gel-modified PMMA microchip. The water contact angle for the sol-gel-modified PMMA was approximately 27.4 degrees compared with approximately 66.3 degrees for the pure PMMA. In addition, the electro-osmotic flow increased from 2.13x10(-4) cm2 V(-1) s(-1) for the native-PMMA channel to 4.86x10(-4) cm2 V(-1) s(-1) for the modified one. The analytical performance of the sol-gel-modified PMMA microchip was demonstrated for the electrophoretic separation of several purines, coupled with amperometric detection. The separation efficiency of uric acid increased to 74,882.3 m(-1) compared with 14,730.5 m(-1) for native-PMMA microchips. The result of this simple modification is a significant improvement in the performance of PMMA for microchip electrophoresis and microfluidic applications.     

Channel wall coating on a poly-(methyl methacrylate) CE microchip by thermal immobilization of a cellulose derivative for size-based protein separation

We demonstrate channel wall coating using a cellulose derivative on a poly-(methyl methacrylate) (PMMA) CE microchip to eliminate EOF disturbing protein separation. The channel walls were modified by preconditioning with a solution containing the cellulose derivative and then thermally evaporating the solution to produce hydrophilic channel walls which prevent adsorption of analytes via a hydrophobic interaction. When the PMMA substrate was coated with the cellulose derivative hydroxypropylmethylcellulose (HPMC) 90SH, the water contact angle on the coated substrate was decreased (up to 15 degrees ) and EOF was significantly suppressed (up to 4.0 x 10(-6) cm2.V(-1)s(-1)). Three proteins (20.5, 68.0, and 114.6 kDa) were successfully separated on the 0.15% HPMC 90SH-coated channel walls with good reproducibility of migration time (RSD <1.75%) and high efficiency (theoretical plate number per meter: 2.62 x 10(5)).     

Robust isocratic liquid chromatographic separation of functional poly(methyl methacrylate)

The separation of telechelic poly(methyl methacrylate) (PMMA) prepolymers based on the number of end-groups under critical liquid chromatography (LC) conditions has been studied using a bare-silica column, which can interact with polar functional groups. The critical solvent compositions for non-functional, mono-functional and bi-functional PMMAs were determined in normal-phase LC using mixtures of acetonitrile and dichloromethane (DCM) of varying composition as the mobile phase. The telechelic prepolymers were successfully separated according to hydroxyl (OH) functionality (with zero, one, or two OH groups, respectively) under the critical conditions, in which fast (5 min), base-line separations were obtained independent of molecular weight. Changing the column temperature, flow rate, and mobile-phase composition within a certain range did not affect the functionality separation. Therefore this isocratic LC separation method is quite robust. Evaporative light-scattering detector (ELSD) calibration curves were used for the quantitative analysis of functional PMMA prepolymers.     

Phase separation studies on poly(vinylidene fluoride) and poly(methyl methacrylate) quenched blends

A combined study by SAXS and DSC on quenched blends of PVDF and PMMA is presented. Attention is focused on the first stage of the phase separation process during annealing that is shown to be mainly determined by the diffusion of the PVDF molecules from the amorphous blend phase towards the crystals growth front. The experimental monomer diffusion constants at T > T_g are compared with those expected theoretically using the approximation of the fast model process and the WLF equation for the relaxation frequency of the monomer. The nature and composition of the crystal interphase are discussed in terms of the SAXS invariant for the whole system and the calorimetric data derived from the T_g transitions 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.     

Ultrathin-layer sodium dodecyl sulfate gel electrophoresis of proteins: effects of gel composition and temperature on the separation of sodium dodecyl sulfate-protein complexes

This paper discusses the effects of gel composition and separation temperature on the migration properties of fluorescein-5-isothiocyanate-labeled protein molecular mass markers (ranging from 20 100 to 205 000 Da) in automated ultrathin-layer sodium dodecyl sulfate (SDS) gel electrophoresis. The separation mechanism with the agarose and composite agarose - linear polyacrylamide, agarose - hydroxyethyl cellulose, and agarose - polyethylene oxide matrices were all found to comply with the Ogston sieving model in the molecular mass range of the protein molecules investigated. Our temperature studies revealed that electrophoretic separation of SDS protein complexes is an activated process and, in pure agarose and in composite agarose hydroxyethyl cellulose and agarose - polyethylene oxide matrices that the separation requires increasing activation energy as a function of the molecular mass of the separated proteins. On the other hand, when linear polyacrylamide was used as composite additive, the activation energy demand of the separation decreased with increasing solute molecular mass. The sensitivity of the laser-induced fluorescent detection of the automated ultrathin-layer electrophoresis system was evaluated by injecting a series of dilutions of the markers and was found to be less than 2.5 ng/band for the fluorophore-labeled protein.     

Dynamic modification of poly(methyl methacrylate) chips using poly(vinyl alcohol) for glycosaminoglycan disaccharide isomer separation

We describe a microchip electrophoresis (MCE) method for the assay of unsaturated disaccharides of chondroitin sulfates, dermatan sulfates, and hyaluronic acid (HA). Poly(vinyl alcohol) (PVA) could be irreversibly adsorbed onto poly(methyl methacrylate) (PMMA) substrates and this approach was applicable for dynamic coating. The characteristics of the PMMA surface with PVA coating were evaluated in terms of the wettability, EOF, and adsorption of 2-aminoacridone (AMAC)-labeled disaccharide. The water contact angle decreased from 73 degrees on a pristine PMMA surface to 37.5 degrees on a PVA-coated surface, indicating that the PVA coating increased hydrophilicity. EOF was reduced approximately twofold and was relatively stable. Scanning electron microscopy and fluorescence microscopy images showed that adsorption of AMAC-labeled disaccharides was dramatically suppressed. Using the PVA coating, baseline separation of two pairs of glycosaminoglycan (GAG) disaccharide isomers, DeltaDi-diS(B)/DeltaDi-diS(D) and DeltaDi-0S/DeltaDi-HA, was achieved in Tris-borate buffer within 130 s by MCE.     

Photodegradation of poly(methyl methacrylate)

The vacuum photodegradation at 30°C. of poly(methyl methacrylate) and copolymers with acrylaldehyde, methacrylaldehyde, and methyl acrylate has been studied. The polymers were examined in the form of expanded films as produced by a freeze-drying technique. At least one molecule of carbon monoxide is evolved for each chain scission. It is concluded that chain scission in poly(methyl methacrylate) is primarily the result of photoinduced aldehyde groups.     

Modification of a poly(methyl methacrylate) injection-molded microchip and its use for high performance analysis of DNA

Inexpensive and permanently modified poly(methyl methacrylate)(PMMA) microchips were fabricated by an injection-molding process. A novel sealing method for plastic microchips at room temperature was introduced. Run-to-run and chip-to-chip reproducibility was good, with relative standard deviation values between 1-3% for the run-to-run and less than 2.1% for the chip-to-chip comparisons. Acrylonitrile-butadiene-styrene (ABS) was used as an additive in PMMA substrates. The proportions of PMMA and ABS were optimized. ABS may be considered as a modifier, which obviously improved some characteristics of the microchip, such as the hydrophilicity and the electro-osmotic flow (EOF). The detection limit of Rhodamine 6G dye for the modified microchip on the home-made microchip analyzer showed a dramatic 100-fold improvement over that for the unmodified PMMA chip. A detection limit of the order of 10(-20) mole has been achieved for each injected psiX-174/HaeIII DNA fragment with the baseline separation between 271 and 281 bp, and fast separation of 11 DNA restriction fragments within 180 seconds. Analysis of a PCR product from the tobacco ACT gene was performed on the modified microchip as an application example.     

Polycation coating poly(dimethylsiloxane) capillary electrophoresis microchip for rapid separation of ascorbic acid and uric acid

A novel method for rapid separation and determination of ascorbic acid and uric acid has been developed with a polycation-modified poly(dimethylsiloxane) (PDMS) microchip under a negative-separation electric field. Just by flushing the microchip with aqueous solutions of the polycations, poly(allylamine) hydrochloride, poly(diallyldimethylammonium chloride) or chitosan could be stably coated on the PDMS microchannel surface, which resulted in a reversed electroosmotic flow and thus the rapid and efficient separation of the two substrates. Factors influencing the separation, including polycation category, buffer solution, detection potential and separation voltage, were investigated and optimized. The cheapness, rapid analysis speed and the successful analysis of human urine make this microsystem attractive for application in clinics. Figure The electropherograms of 100 μ/mL AA and UA in (1) PAH, (2) PDDA, (3) Chitosan modified PDMS microchannels and native PDMS microchip (4).     

Co-nonsolvent systems for poly(ϵ-caprolactone) and poly(methyl methacrylate)

The cloud points for the co-nonsolvent systems (i) pyridine (PY) + formic acid (FA) towards poly(?-caprolactone) (PCL) or poly(methyl methacrylate) (PMMA) and (ii) pyridine + acetic acid (AA) towards PCL have been experimentally determined as a function of solvent composition at fixed polymer concentrations. Heats of mixing for the (FA + PY) system have also been measured. The single liquid approximation model correctly predicts for the (PY + FA) system the solvent composition at which the solvent power of the mixture is the poorest, provided it is recognized that the solvent system is comprised of pyridinium formate (PYFA) as one component and either PY or FA (depending on which is present in excess over 1:1 mole ratio) as the other. With the other co-nonsolvent system, the prediction is not as good. A possible reason for the discrepancy has been given.     

Dynamics of adsorbed poly(methyl acrylate) and poly(methyl methacrylate) on silica

Deuterium NMR and modulated differential scanning calorimetry (MDSC) were used to probe the behavior of ultrathin adsorbed poly(methyl acrylate) (PMA). The spectra for the bulk methyl-labeled PMA-d₃ were consistent with the motions of the polymer segments being spatially homogeneous. For the polymers adsorbed on silica, multicomponent line shapes were observed. The segmental mobility of the surface polymers increased with increased adsorbed amounts. In contrast to the behavior of the polymers in bulk, the adsorbed lower-molecular-mass PMA-d₃ was less mobile than the adsorbed high-molecular-mass polymer. The presence of a polymer overlayer was sufficient to suppress the enhanced mobility of the more-mobile segments of the adsorbed (inner) polymer. MDSC studies on adsorbed poly(methyl methacrylate) showed that the glass-transition temperature of the thin polymer films increased and broadened compared to the behavior of the polymer in bulk. The presence of a motional gradient with the less-mobile segments near the solid-polymer interface and the more-mobile segments near the polymer-air interface was consistent with the experimental observations.     

Compatibilization of blends of polybutadiene and poly(methyl methacrylate) with poly(butadiene-block-methyl methacrylate)

Compatibilization of blends of polybutadiene and poly(methyl methacrylate) with butadiene-methyl methacrylate diblock copolymers has been investigated by transmission electron microscopy. When the diblock copolymers are added to the blends, the size of PB particles decreases and their size distribution gets narrower. In PB/PMMA7.6K blends with P(B-b-MMA)25.2K as a compatibilizer, most of micelles exist in the PMMA phase. However, using P(B-b-MMA)38K as a compatibilizer, the micellar aggregation exists in PB particles besides that existing in the PMMA phase. The core of a micelle in the PMMA phase is about 10 nm. In this article the influences of temperature and homo-PMMA molecular weight on compatibilization were also examined. At a high temperature PB particles in blends tend to agglomerate into bigger particles. When the molecular weight of PMMA is close to that of the corresponding block of the copolymer, the best compatibilization result would be achieved. © 1998 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 36 : 85–93, 1998     

Effect of crosslinking on porous poly(methyl methacrylate) produced by phase separation

Porous polymethyl methacrylate scaffolds were produced by phase separation during polymerisation in solution, using ethanol as solvent, with monomer/ethanol weight ratios from 80/20 up to 20/80 and different ethylenglycol dimethacrylate contents (1, 5 and 10 wt%) as crosslinker agent. For ethanol weight ratios equal to or lower than 50 wt%, the material presents a homogeneous distribution of dispersed pores. For higher ethanol contents, a highly interconnected porous structure is obtained. The transition from one type of morphology to the other can be also controlled with the amount of crosslinker added in the reactive mixture. Bulk polymethyl methacrylate samples (non-porous) with the same crosslinking densities were also synthesised as reference. The effect of crosslinker is studied by porosity measurements, scanning electron microscopy, dynamic-mechanical spectroscopy and differential scanning calorimetry.     

Thermoluminescence of irradiated poly(methyl methacrylate)

Thermoluminescence of poly(methyl methacrylate) (PMMA) irradiated with x rays, has been studied in the temperature range 100 to 460°K. Two glow peaks with maxima at 136 and 368°K have been observed. These are analyzed by three methods and the results are compared. Both curves obey second order kinetics and correspond to activation energies of 0.17 and 0.88 eV, respectively. It is possible to identify the centers responsible for the two peaks by correlation with electron spin resonance and optical data obtained for the same samples irradiated under the same conditions. Spectral studies of the emission show that the low temperature peak has its maximum at 365 nm while the high temperature peak has its maximum at 480 nm.