Nonconventional synthesis and characterization of ultrahigh-molar-mass polyacrylamides

In spite of the significant progresses in the field of replaceable sieving matrices for separating DNA in capillary electrophoresis (CE), an intense research activity is still going on to improve the separation of large size DNA sequencing fragments. There are evidences, both from experimental and theoretical sides that the resolution of these fragments, at the single base, requires the use of sieving matrices comprised of long chain linear polymers. In the separation of DNA fragments by CE are of upmost importance: (i) the complete solubility of the polymer, (ii) the linearity of the chain, (iii) the achievement of ultrahigh viscosity in dilute solutions. The aim of this work is the synthesis of ultrahigh-molecular-weight polymers which possess the three requirements mentioned above by employing a nonconventional method. We demonstrate that the sieving performance of polyacrylamide is directly correlated to its intrinsic viscosity.     

毛细管电泳无胶筛分介质分离DNA的机理

快速、高效而灵敏的分离技术对于DNA的分析是至关重要的。使用无胶筛分介质的毛细管电泳是最重要的DNA分离技术之一,通常使用无交联的高分子溶液作为无胶筛分介质。本文在介绍高分子溶液理论的基础上,综述了DNA在毛细管电泳无胶筛分介质(缠结溶液和稀溶液)中的分离机理,主要包括Ogston筛分模型、各种修正的爬行模型、瞬态缠结偶合机理及其改进机理等。     

Reduced viscosity polymer matrices for microchip electrophoresis of double-stranded DNA

On a polymethylmethacrylate (PMMA) microchip, double-stranded DNA fragments with a wide size range from 50 bp to 20 kbp were separated by two polymer solutions. One was a hydroxypropylmethylcellulose-4000 (HPMC-4000) solution of 1.3% (w/v) to separate fragments below 590 bp, and another was a mixed four molecular weight poly(ethylene oxide) solution at a total concentration of 0.1% to separate fragments above 520 bp. The widths at half height (wh) of the fragments had a good relationship with their migration times (tR) in both polymer solutions. Such a relationship was suitable for obtaining the wh values of unresolved peaks, calculating the resolution of two adjacent fragments, and optimizing microchip separation matrices. Based on the relativity, a low viscosity medium containing 2% HPMC-50 and 8% glucose was optimized for high-performance separation of a phiX174 HaeIII restriction fragment digest.     

A DNA sieving matrix with thermally tunable mesh size

We present a "proof-of-concept" study showing that a blend of thermo-responsive and nonthermo-responsive polymers can be used to create a DNA sieving matrix with a thermally tunable mesh size, or "dynamic porosity". Various blends of two well-studied sieving polymers for CE, including hydroxypropylcellulose (HPC), a thermo-responsive polymer, and hydroxyethylcellulose (HEC), a nonthermo-responsive polymer, were used to separate a double-stranded DNA restriction digest (Phi X174-HaeIII). HPC exhibits a volume-phase transition in aqueous solution which results in a collapse in polymer coil volume at approximately 39 degrees C. Utilizing a blend of HPC and HEC in a ratio of 1:5 by weight, we investigated the effects of changing mesh size on DNA separation, as controlled by temperature. High-resolution DNA separations were obtained with the blended matrix at temperatures ranging from 25 degrees C to 38 degrees C. We evaluated changes in the selectivity of DNA separation with increasing temperature for certain pairs of small and large fragments. A pure HEC (nonthermo-responsive) matrix was used over the same temperature range as a negative control. In the blended matrix, we observe a maximum in selectivity at approximately 31 degrees C for small DNA, while a significant increase in the selectivity of large-DNA separation occurs at approximately 36 degrees C as the polymer mesh "opens". We also demonstrate, through a temperature ramping experiment, that this matrix can be utilized to obtain high-resolution separation of both small and large DNA fragments simultaneously in a single CE run. Blended polymer matrices with "dynamic porosity" have the potential to provide enhanced genomic analysis by capillary array or microchip electrophoresis in microfluidic devices with advanced temperature control.     

Separation of DNA fragments in hydroxylated poly(dimethylacrylamide) copolymers

The novel polymer matrices reported here are low-viscosity sieving media for DNA capillary electrophoresis. This new family of matrices comprises copolymers of N,N-dimethylacrylamide with different monomers which increase polymer hydrophilicity. All these new copolymers self-coat on fused-silica capillaries. Resolution, peak spacing and peak width were the parameters taken into account to assess the influence of polymer structure on separation selectivity and efficiency. This work demonstrates that the performance of polydimethylacrylamide (PDMA) can be improved through copolymerization with hydrophilic monomers. The improvement is related to the efficiency parameter. The new copolymers, due to their low viscosity high sieving capacity and ability to suppress EOF, represent a better alternative to PDMA and are suitable replaceable matrices for capillary and microchip electrophoresis.     

Evaluation of sieving matrices used to separate alleles by cycling temperature capillary electrophoresis

Denaturing CE (DCE) is a powerful tool for analysis of DNA variation. The development of commercial multi-CE instruments allows large-scale studies of DNA variation (many samples and many fragments). However, the cost of consumables like capillary arrays and sieving matrix might limit the use of DCE in such studies. Thus, we have tested 72 different in-house formulated sieving matrices' ability to suppress EOF and separate PCR-amplified alleles with the DCE variant, cycling temperature CE (CTCE). The data herein demonstrate that alleles can be baseline-separated by use of PVP and poly(N,N-dimethyl acrylamide) polymers at various percentages and pH. Allele separation by CTCE is matrix-independent and consequently applicable to any capillary instrument used for DNA separation. Formulation of sieving matrix for CTCE was done by dissolving appropriate amount of polymer powder into the running buffers. Allele separation was observed at different pH (7.5-8.5), concentrations and molecular size of the polymer, without compromising the separation and reproducibility. Finally, the cost reduction of homemade matrices is more than 1000-fold as compared to commercial sieving matrices.     

Nanomaterials and chip-based nanostructures for capillary electrophoretic separations of DNA

Capillary electrophoresis (CE) and microchip capillary electrophoresis (MCE) using polymer solutions are two of the most powerful techniques for the analysis of DNA. Problems, such as the difficulty of filling polymer solution to small separation channels, recovering DNA, and narrow separation size ranges, have put a pressure on developing new techniques for DNA analysis. In this review, we deal with DNA separation using chip-based nanostructures and nanomaterials in CE and MCE. On the basis of the dependence of the mobility of DNA molecules on the size and shape of nanostructures, several unique chip-based devices have been developed for the separation of DNA, particularly for long DNA molecules. Unlike conventional CE and MCE methods, sieving matrices are not required when using nanostructures. Filling extremely low-viscosity nanomaterials in the presence and absence of polymer solutions to small separation channels is an alternative for the separations of DNA from several base pairs (bp) to tens kbp. The advantages and shortages of the use of nanostructured devices and nanomaterials for DNA separation are carefully addressed with respect to speed, resolution, reproducibility, costs, and operation.     

Sieving matrices in capillary electrophoresis: inflection slope and double reciprocal plot

We propose the use of a double reciprocal plot of the inflection slope and the concentration of a sieving polymer to compare sieving properties of polymers regardless of their concentration and to permit selection of appropriate sieving materials. Using this plot, the lines extrapolating the experimental values intersect the ordinate at the value corresponding to the inflection slope at the infinite concentration of the sieving polymer and the abscissa at the value characteristic for the given polymer. The latter corresponds to the polymer concentration at which the inflection slope equals half the inflection slope at an infinite polymer concentration. It is inversely proportional to intrinsic viscosity and this makes intrinsic viscosity an important physicochemical constant suitable for selection of new potent sieving polymers.     

Investigation of Metal Ion Removal Selectivity Properties of the Modified Polyacrylamide Hydrogels Prepared by Transamidation and Hofmann Reactions

Modified crosslinked polyacrylamides having different functional groups prepared by transamidation reaction in aqueous and non‐aqueous medium and by Hofmann reaction were used as chelating agents for removal of Cu(II), Cd(II) and Pb(II) ions from aqueous solutions at different pH values. Under non‐competitive conditions, polymers adsorbed different amounts of metal ions, depending on their functional groups and swelling abilities. The metal ion adsorption capacities of polymers changed between 0.11–1.71 mmol/g polymer. Under competitive conditions, while the polymers having mainly secondary amine groups were highly selective for Cu(II) ions (99.4%), those having mainly secondary amide and carboxylate groups have shown high selectivity towards Pb(II) ions (99.5%). The selectivity towards Cu(II) ion decreased and Pb(II) ion selectivity increased by the decrease of the pH of the solutions. The high initial adsorption rate (<10 min) suggests that the adsorption occurs mainly on the polymer surface. A regeneration procedure by treatment with dilute HCl solution showed that the modified polymers could be used several times without loss of their adsorption capacities.     

Effects of novel quasi-interpenetrating network/gold nanoparticles composite matrices on DNA sequencing performances by CE

Gold nanoparticles (GNPs) with particle sizes of about 20, 40, and 60 nm were prepared and added into a quasi-interpenetrating network (quasi-IPN) composed of linear polyacrylamide (LPA) with different viscosity-average molecular masses of 1.5, 3.3, and 6.5 MDa and poly-N,N-dimethylacrylamide (PDMA) to form polymer/metal composite matrices, respectively. These novel matrices could improve ssDNA sequencing performances due to interactions between GNPs and polymer chains and the formation of physical cross-linking points as demonstrated by intrinsic viscosities and glass transition temperatures. The effects of the parameters in relation to quasi-IPN/GNPs matrices, such as GNP contents, GNP particle sizes, LPA molecular masses, and solution concentrations, on ssDNA sequencing performances were studied. In the presence of GNPs, the separation had the advantages of high resolution, speediness, excellent reproducibility, long shelf life and easy automation. Therefore, less viscous matrix solutions (with moderate size GNPs) due to lower solution concentration and lower-molecular-mass LPA could be used to replace more viscous solutions (without GNPs) due to higher solution concentration or higher-molecular-mass LPA to separate DNA, while the sieving performances were approximate even higher, which helped to achieve full automation especial for capillary array electrophoresis (CAE) and microchip electrophoresis (MCE).     

Sieving mechanisms in polymeric matrices

A critical review of the existing theoretical models and experimental evidences for sieving mechanisms during separation of macromolecules, paying particular attention to capillary electrophoresis applications is presented. Gel models (Ogston and reptation) have been successfully applied to highly entangled polymer solutions, where fast and efficient separations can occur. In order to account for the DNA/polymers collision-interaction mechanisms during separation in dilute solutions - characterized by a poorer resolution -, approximated analytical models have been developed. An insight in the mechanism regulating the intermediate case of moderately entangled polymer solutions, for low fields and concentrations of small multiples of the overlap concentration c*, is given by the constraint release approach. This model proposes an upper limit of size separation, increasing with matrix concentration and molecular mass. Finally, the coupling between the reptative motion of the analytes and the effect of matrix constraint release very likely plays a fundamental role in the separation mechanism and requires therefore further and deeper investigation, both theoretically and experimentally.     

Size separation of sodium dodecyl sulfate–proteins by capillary electrophoresis in dilute and ultra-dilute dextran solutions

SDS capillary gel electrophoresis is a widely used in the biopharma and the biomedical fields for rapid size separation of proteins. However, very limited information is available on the use of dilute and ultra-dilute sieving matrices for SDS–protein analysis. Here, background electrolytes (BGEs) containing 1%–0% dextran were used in borate-based BGE to separate a protein sizing ladder (PSL) ≤225 kDa and the intact and subunit forms of a therapeutic monoclonal antibody (mAb). The separation performance for the PSL and mAb components differed significantly with decreasing dextran concentration. Ferguson and reptation plots were used to elucidate the separation mechanism. Highly diluted dextran solutions resulted in linear Ferguson plots for both solute types (cf. Ogston theory) in spite of this model assumes a rigid pore structure, thus cannot describe the separation mechanism in ultra-dilute polymer solutions with no reticulations. The saddle differences between the resolution of the PSL and the intact/subunit mAb forms in ultra-dilute dextran-borate matrices suggested the importance of shape selectivity, manifested by the adequate separation of the SDS covered intact as well as light and heavy chain subunits of the therapeutic mAb even at zero dextran concentration.     

Low viscosity DNA sieving matrices for capillary electrophoresis

The rapid development of DNA capillary electrophoresis (CE) technology has increased the demand of new low viscosity sieving matrices with high separation capacity. The high throughput, resolution and automatic operation of CE systems have stimulated the application of the technique to different kinds of DNA analysis, including DNA sequencing, separation of restriction fragments, PCR products and synthetic oligonucleotides. In addition specific methods for PCR-based mutation assays for the study of known and unknown point mutations have been developed for use in CE. The key component for a large scale application of CE to DNA analysis is the availability of appropriate sieving matrices. This article gives an overview of the linear polymers used as DNA separation matrices with particular emphasis on the polymers that combine high sieving capacity, low viscosity and chemical resistance.     

Peculiarities of flow and viscoelastic properties of solutions of polymers with a narrow molecular-weight distribution

The concepts developed for flexible-chain linear high-molecular-weight polymers of narrow molecular weight distribution, according to which transition from the fluid to the highelastic state at increased shear rates is accompanied by loss of fluidity, have been extended to concentrated solutions of these polymers. The change of principal viscoelastic characteristics, in a concentration range which can be associated with the effect of formation of an entanglement network in polymer solution, is considered. On the basis of the parameters characterizing the viscous properties of dilute polymer solutions (the intrinsic viscosity and the Martin constant), a generalized representation of the dependence of the initial viscosity of polybutadiene solutions on concentration over the entire range up to bulk polymer has been derived.     

Microchannel DNA sequencing matrices with switchable viscosities

We review the variety of thermo-responsive and shear-responsive polymer solutions with "switchable" viscosities that have been proposed for application as DNA sequencing matrices for capillary and microfluidic chip electrophoresis. Generally, highly entangled polymer solutions of high-molar mass polymers are necessary for the attainment of long DNA sequencing read lengths (> 500 bases) with short analysis times (< 3 h). However, these entangled polymer matrices create practical difficulties for microchannel electrophoresis with their extremely high viscosities, necessitating high-pressure loading into capillaries or chips. Shear-responsive (shear-thinning) polymer matrices exhibit a rapid drop in viscosity as the applied shear force is increased, but still require a high initial pressure to initiate flow of the solution into a microchannel. Polymer matrices designed to have thermo-responsive properties display either a lowered (thermo-thinning) or raised (thermo-thickening) viscosity as the temperature of the solution is elevated. These properties are generally designed into the polymers by the incorporation of moderately hydrophobic groups in some part of the polymer structure, which either phase-separate or hydrophobically aggregate at higher temperatures. In their low-viscosity states, these matrices that allow rapid loading of capillary or chip microchannels under low applied pressure. The primary goal of work in this area is to design polymer matrices that exhibit this responsive behavior and hence easy microchannel loading, without a reduction in DNA separation performance compared to conventional matrices. While good progress has been made, thermo-responsive matrices have yet to offer sequencing performance as good as nonthermo-responsive networks. The challenge remains to accomplish this goal through the innovative design of novel polymer structures.     

Ultrafast, efficient separations of large-sized dsDNA in a blended polymer matrix by microfluidic chip electrophoresis: a design of experiments approach

Double-stranded (ds) DNA fragments over a wide size range were successfully separated in blended polymer matrices by microfluidic chip electrophoresis. Novel blended polymer matrices composed of two types of polymers with three different molar masses were developed to provide improved separations of large dsDNA without negatively impacting the separation of small dsDNA. Hydroxyethyl celluloses with average molar masses of ～27 kDa and ～1 MDa were blended with a second class of polymer, high-molar mass (～7 MDa) linear polyacrylamide. Fast and highly efficient separations of commercially available DNA ladders were achieved on a borosilicate glass microchip. A distinct separation of a 1-kb DNA extension ladder (200-40,000 bp) was completed in 2 min. An orthogonal design of experiments was used to optimize experimental parameters for DNA separations over a wide size range. We find that the two dominant factors are the applied electric field strength and the inclusion of a high concentration of low-molar mass polymer in the matrix solution. These two factors exerted different effects on the separations of small dsDNA fragments below 1 kbp, medium dsDNA fragments between 1 and 10 kbp, and large dsDNA fragments above 10 kbp.     

Capillary electrophoresis of RNA in dilute and semidilute polymer solutions

We report separations of RNA molecules (281-6583 nucleotides) by capillary electrophoresis in dilute and semidilute solutions of aqueous hydroxyethylcellulose (HEC) ether in varying buffers. RNA mobility and peak band widths are examined under both nondenaturing and also denaturing conditions. From studies of sieving polymer concentration and chain length, it is found that good separations can be obtained in semidilute solutions as well as in dilute solutions. The dependence of RNA mobility on its chain length is consistent with separation by a similar to transient entanglement mechanism in dilute solutions. In semidilute entangled solutions the separation proceeds by segmental motion.     

On-channel base stacking in microchip capillary gel electrophoresis for high-sensitivity DNA fragment analysis

We evaluated a novel strategy for high-sensitivity DNA fragment analysis in a conventional glass double-T microfluidic chip. The microchip allows for a DNA on-channel concentration based on base stacking (BS) with a microchip capillary gel electrophoretic (MCGE) separation step in a poly(vinylpyrrolidone) (PVP) sieving matrix. Depending if low conductivity caused a neutralization reaction between the hydroxide ions and the run buffer component Tris+, the stacking of DNA fragments were processed in the microchip. Compared to a conventional MCGE separation with a normal electrokinetic injection, the peak heights of 50-2650-base pair (bp) DNA fragments on the MCGE-BS separation were increased 3.9-8.0-fold. When we applied the MCGE-BS method to the analysis of a clinical sample of bovine theileria after PCR reaction, the peak height intensity of the amplified 816-bp DNA fragment from the 18S rRNA of T. buffeli was enhanced 7.0-fold compared to that of the normal injection method.     

Electrophoretic migration of proteins in semidilute polymer solutions

We present a systematic study of the electrophoretic migration of 10-200 kDa protein fragments in dilute-polymer solutions using microfluidic chips. The electrophoretic mobility and dispersion of protein samples were measured in a series of monodisperse polydimethylacrylamide (PDMA) polymers of different molecular masses (243, 443, and 764 kDa, polydispersivity index <2) of varying concentration. The polymer solutions were characterized using rheometry. Prior to loading onto the microchip, the polymer solution was mixed with known concentrations of SDS (SDS) surfactant and a staining dye. SDS-denatured protein samples were electrokinetically injected, separated, and detected in the microchip using electric fields ranging from 100 to 300 V/cm. Our results show that the electrophoretic mobility of protein fragments decreases exponentially with the concentration c of the polymer solution. The mobility was found to decrease logarithmically with the molecular weight of the protein fragment. In addition, the mobility was found to be independent of the electric field in the separation channel. The dispersion is relatively independent of polymer concentration and it first increases with protein size and then decreases with a maximum at about 45 kDa. The resolution power of the device decreases with concentration of the PDMA solution but it is always better than 10% of the protein size. The protein migration does not seem to correspond to the Ogston or the reptation models. A semiempirical expression for mobility given by van Winkle fits the data very well.     

Separation of transfer RNA and 5S ribosomal RNA using capillary electrophoresis

Capillary gel electrophoresis and capillary electrophoresis using entangled polymer solutions was investigated for their applicability for the separation of low-molecular-mass RNAs (transfer RNA and 5S ribosomal RNA), with a size range of 70–135 nucleotides, from bacteria. Cross-linked polyacrylamide gel-filled capillaries (3 and 5%) were used for capillary gel electrophoresis. Good resolution was obtained suing gel-filled capillaries only for small tRNAs with lengths to 79 nucleotides, larger tRNAs and 5S rRNA could not be resolved using this method. Buffers containing sieving additives were employed to improve separations of RNA by capillary electrophoresis using entangled polymer solutions. The use of linear sieving polymers in buffers resolved 5S rRNA and tRNAs, even when they possessed only different secondary structure or small differences in length (1–5 nucleotides).