Polymeric matrices for DNA sequencing by capillary electrophoresis

We review the wide range of polymeric materials that have been employed for DNA sequencing separations by capillary electrophoresis. Intensive research in the area has converged in showing that highly entangled solutions of hydrophilic, high molar mass polymers are required to achieve high DNA separation efficiency and long read length, system attributes that are particularly important for genomic sequencing. The extent of DNA-polymer interactions, as well as the robustness of the entangled polymer network, greatly influence the performance of a given polymer matrix for DNA separation. Further fundamental research in the field of polymer physics and chemistry is needed to elucidate the specific mechanisms by which DNA is separated in dynamic, uncross-linked polymer networks.     

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。     

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.     

Polymer solutions and entropic-based systems for double-stranded DNA capillary electrophoresis and microchip electrophoresis

We give an overview of recent development of low-viscosity polymer solutions and entropic trapping networks for double-stranded DNA (dsDNA) separations by conventional capillary electrophoresis and microchip electrophoresis. Theoretical models for describing separation mechanisms, commonly used noncross-linked polymer solutions, thermoresponsive (viscosity-adjustable) polymer solutions, and novel entropic trapping networks are included. The thermoresponsive polymer solutions can be loaded at one temperature into microchannels at lower viscosities, and used in separation at another temperature at entanglement threshold concentrations and higher viscosities. The entropic-based separations use only arrays of regular obstacles acting as size-separations and do not need viscous polymer solutions. These progresses have potential in integration to automated capillary and microfluidic chip systems, enabling better reusability of separation microchannels, much shorter DNA separation times, and higher reproducibility due to less matrix degradation.     

Electrophoretic migration behavior of DNA fragments in polymer solution

A newly developed polymer coil shrinking theory is described and compared with the existing entangled solution theory to explain electrophoretic migration behaviour of DNA in hydroxypropylmethylcellulose (HPMC) polymer solution in buffer containing 100 mM tris(hydroxymethyl)aminomethane 100 mM boric acid, 2 mM ethylenediaminetetraacetic acid at pH 8.3. The polymer coil shrinking theory gave a better model to explain the results obtained. The polymer coil shrinking concentration, Cs, was found to be 0.305% and the uniform entangled concentration, C+, 0.806%. The existence of three regions (the dilute, semidilute, and concentrated solution) at different polymer concentrations enables a better understanding of the system to guide the selection of the best conditions to separate DNA fragments. For separating large fragments (700/ 800 bp), dilute solutions (HPMC < 0.3%) should be used to achieve a short migration time (10 min). For small fragments (200/300 bp), concentrated solutions are preferred to obtain constant resolution and uniform separation. The best resolution is 0.6% HPMC due to a combined interaction of the polymer coils and the entangled structure. The possibility of DNA separation in semidilute solution is often neglected and the present results indicate that this region has a promising potential for analytical separation of DNA fragments.     

Size-based separation of synthetic polyelectrolytes in entangled polymer solution capillary electrophoresis: the effect of binary mixtures of separating polymers differing in molecular mass

The influence on the electrophoretic behavior of polystyrenesulfonates of the percentage of high-molecular-mass chains in an entangled poly(ethylene oxide) solution having a bimodal molecular mass distribution has been investigated and compared with the results obtained for similar solutions of unimodal molecular mass distribution. The comparisons between the different separating polymer solutions were made at a constant total mass concentration, so as to keep constant the mesh size and to highlight the sole effect of the network dynamics. The use of binary polymer mixtures of two different molecular masses but of same nature can be a convenient alternative to modulate the dynamics of the network and the viscosity of the separating medium. A 20-30% content of high-molecular-mass chains in an entangled poly(ethylene oxide) solution having a binary molecular mass distribution appears to be a good compromise for a moderate viscosity and a good separation selectivity in comparison with a solution containing only chains of high molecular mass at the same concentration.     

Mathematical model for DNA separation by capillary electrophoresis in entangled polymer solutions

A mathematical model of DNA separation by capillary electrophoresis in entangled polymer solution is presented. The mechanism is modeled as a DNA molecule moving through transient pores formed in polymer solutions and colliding with blobs of polymer molecules encountered during migration. By taking account of the average retardation time (t(c)) of DNA-blob collision and calculating the total collision number (N(c)), a quantitative mathematical equation was reported, leading to predictions for the DNA mobility as a function of the experimental conditions like the size of DNA, the polymer concentration and the electric field strength. For DNA fragments in frequent size range, the initial experimental data agree well with the model. The DNA shape function (f(E)) was suggested and then discussed by the experimental data. The relationship between f(E) and electric field strength E was empirically estimated. Then, the average retardation time t(c) was obtained as about (2 approximately 3)x10(-6)s in linear polyacrylamide (LPA) and hydroxyethylcellulose (HEC) solution.     

Strain Hardening and Strain Softening of Reversibly Cross-linked Supramolecular Polymer Networks

The large amplitude oscillatory shear behavior of metallo-supramolecular polymer networks formed by adding bis-Pd(II) cross-linkers to poly(4-vinylpyridine) (PVP) in dimethyl sulfoxide (DMSO) solution is reported. The influence of scanning frequency, dissociation rate of cross-linkers, concentration of cross-linkers, and concentration of PVP solution on the large amplitude oscillatory shear behavior is explored. In semidilute unentangled PVP solutions, above a critical scanning frequency, strain hardening of both storage moduli and loss moduli is observed. In the semidilute entangled regime of PVP solution, however, strain softening is observed for samples with faster cross-linkers (k(d) ～ 1450 s(-1)), whereas strain hardening is observed for samples with slower cross-linkers (k(d) ～ 17 s(-1)). The mechanism of strain hardening is attributed primarily to a strain-induced increase in the number of elastically active chains, with possible contributions from non-Gaussian stretching of polymer chains at strains approaching network fracture. The divergent strain softening of samples with faster cross-linkers in semidilute entangled PVP solutions, relative to the strain hardening of samples with slower cross-linkers, is consistent with observed shear thinning/shear thickening behavior reported previously and is attributed to the fact that the average time that a cross-linker remains detached is too short to permit the local relaxation of polymer chain segments that is necessary for a net conversion of elastically inactive to elastically active cross-linkers. These and other observations paint a picture in which strain softening and shear thinning arise from the same set of molecular mechanisms, conceptually uniting the two nonlinear responses for this system.     

缠结聚合物溶液毛细管电泳

从缠结聚合物溶液理论及其电泳迁移理论对缠结聚合物溶液毛细管电泳的现状给予综述，并就缠结聚合物溶液毛细和电泳在ＤＮＡ，蛋白质等大分子分离方面的应用进行了概述。     

Modeling the solid-like behavior of entangled polymer nanocomposites at low frequency regimes

A theory for the linear viscoelastic behavior of entangled polymeric liquids reinforced with non-aggregated colloidal nanoparticles is presented. Composites with low filler concentration and strong polymer-particle interaction are considered. A fraction of entangled chains is assumed to be reversibly adsorbed on the surface of fillers, due to the affinity between the polymer molecules in the matrix and dispersed filler particles. The relaxation of the system is analyzed by the combination of stress relaxation functions for free and adsorbed polymer chains. It is demonstrated that the emergence of solid-like behavior at low frequency regimes, is due to the significant slow down in relaxation of adsorbed chains. Fitting the model predictions with relevant experimental data indicates that while the effect of constraint release should be considered to obtain a reasonable estimation of neat polymer behavior, the linear combination of stress relaxation functions of free and adsorbed chains (i.e., no thermal constraint release) leads to a better agreement with experimental data of filled systems.     

DNA separation by microchip electrophoresis using low-viscosity hydroxypropylmethylcellulose-50 solutions enhanced by polyhydroxy compounds

Low-viscosity polymer solutions have potential for double-stranded (ds) DNA separations in micrototal analysis systems (micro-TAS). In this paper, we report dilute, low-viscosity hydroxypropylmethylcellulose-50 (HPMC-50, 11.5 kDa) solutions containing polyhydroxy additives as separation media. Predominant operational variables, such as applied electric field strength, fluorescent intercalator (YOPro-1) concentration, polymer concentration, and additive concentration, are thoroughly investigated. Fast (within 170 s) and excellent separation of DNA restriction fragments ranging in size from 72 to 1353 base pairs (bp) is achieved in a 30 mm length channel of polymethylmethacrylate (PMMA) microchips at an electric field strength of 300 V/cm, by introducing 8% mannitol, 8% glucose or 10% glycerol additives into a 2% HPMC-50/1 x Tris-borate-EDTA (TBE) solution. The low-viscosity (40 cP) matrix formulation provides both coating of the microchannels and separation of DNA in one step. The performance in the solution surpasses that in highly concentrated HPMC-50 solution. In addition, separation using 1xTris-EDTA buffer in the 2% HPMC-50 matrix containing polyhydroxy additives also exhibits a notably increased performance. This is presumably due to formation of hydrogen-bonding interactions of polyhydroxy additives with HPMC-50 matrix and DNA so as to increase the coupling interactions between matrix and DNA molecules during electrophoresis. The result reflects that boric acid is not a prerequisite in polyhydroxy-enhanced HPMC-50 solution for separation.     

Acoustic absorption and dynamic compressibility studies of dilute and concentrated solutions of polystyrene in toluene and cyclohexane

Acoustic absorption and adiabatic compressibility measurements are reported on solutions of polystyrene (M_n = 89,000) in toluene and cyclohexane. The data in toluene cover a temperature range from 293 to 343°K and a concentration range of 10–400 Kg m^?3 (1–40 wt%). The dependence of acoustic absorption on concentration was found to be linear up to 100 kg m^?3, which corresponds to the concentration at which polymer–polymer interactions cause significant changes in the specific viscosity-concentration relationship. Up to 200 kg m^?3 the data could be fitted to computations based on an artificial separation of the dispersion into contributions from viscoelastic and segmental processes, using parameters obtained from a study of narrow molecular weight distribution samples at 25 kg m^?3. However, neither approach was capable of describing dispersions in the 300, 400 kg m^?3 solutions. The modification of the relaxation spectrum observed at the highest concentrations is ascribed to volume and entropy changes associated with alterations of the local environment around a segment of the polymer chain. These changes have their origin in interchain penetration and polymer–polymer contacts, and indicate that ‘entanglement’ is primarily entropic in effect. The adiabatic compressibility exhibited similar deviations from a simple concentration dependence, and allowed estimation of an incompressible volume increment associated with polymer–polymer interactions in the high-concentration entangled matrix. However, the adiabatic compressibilities of solutions of polystyrene, 10–15 kg m^?3, in cyclohexane showed no deviations from simple behavior in the region of the theta temperature. Measurements of the adiabatic compressibility of polystyrene in mixtures of cyclohexane-toluene have been used to obtain the relative magnitude of solvent and polymer contributions to the excess compressibility.     

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.     

Interdiffusion of solvent into glassy polymer films: a molecular dynamics study

Large scale molecular dynamics and grand canonical Monte Carlo simulation techniques are used to study the behavior of the interdiffusion of a solvent into an entangled polymer matrix as the state of the polymer changes from a melt to a glass. The weight gain by the polymer increases with time t as t(1/2) in agreement with Fickian diffusion for all cases studied, although the diffusivity is found to be strongly concentration dependent especially as one approaches the glass transition temperature of the polymer. The diffusivity as a function of solvent concentration determined using the one-dimensional Fick's model of the diffusion equation is compared to the diffusivity calculated using the Darken equation from simulations of equilibrated solvent-polymer solutions. The diffusivity calculated using these two different approaches are in good agreement. The behavior of the diffusivity strongly depends on the state of the polymer and is related to the shape of the solvent concentration profile.     

Separation studies of hydrazine from aqueous solutions by pervaporation

Separation of hydrazine from aqueous solutions with ethylcellulose membranes has been investigated by using the pervaporation technique. The effect of membrane thickness, concentration polarization, and feed concentration on flux and selectivity were evaluated. A separation mechanism is proposed based on the measurements of sorption, and diffusion coefficients, and estimations of Flory–Huggins interaction parameters and Hansen's solubility parameter. States of water, hydrazine, and hydrazine hydrate are explained with DSC spectra. The specific interaction sites in ethylcellulose matrix where the solvent interacts extensively with the polymer have been identified by FTIR analysis. © 1999 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 37: 1969–1980, 1999     

Metal ions diffusion through polymeric matrices: A total reflection X-ray fluorescence study

This work proposes the use of X-ray fluorescence with total reflection geometry to explore the metal ions transport in aqueous hydrophilic polymer solutions. It is centered in the study of polymer concentration influence on ion diffusion. This subject is relevant to various and diverse applications, such as drug controlled release, microbiologic corrosion protection and enhanced oil recovery. It is anticipated that diffusion is influenced by various factors in these systems, including those specific to the diffusing species, such as charge, shape, molecular size, and those related to the structural complexity of the matrix as well as any specific interaction between the diffusing species and the matrix. The diffusion of nitrate salts of Ba and Mn (same charge, different hydrodynamic radii) through water-swollen polymeric solutions and gels in the 0.01% to 1% concentration ranges was investigated. The measurements of the metal concentration were performed by TXRF analysis using the scattered radiation by the sample as internal standard. Results are discussed according to different physical models for solute diffusion in polymeric solutions.     

Kinetics and equilibria associated with the absorption and desorption of water and lithium chloride in an ethylene—vinyl alcohol copolymer

Sorption and desorption equilibria and kinetics for LiCl and H₂O in an ethylene—vinyl alcohol copolymer film containing 70 mole percent vinyl alcohol were investigated at 25°C. The swelling behavior of water in the polymer was characterized by vapor and liquid sorption experiments over a range of water activities. p]The effects of LiCl content on the water sorption kinetics and equilibria in the films are presented and discussed. The kinetics and mechanism of LiCl sorption have also been studied. The amount of salt sorbed into the polymeric films increases linearly with the salt concentration in the external aqueous solutions. Both the rate and the amount of sorbed water increase significantly as the LiCl content increases. p]The desorption of LiCl, previously sorbed into the polymer, was characterized for different salt loadings. The rate of fractional salt release is independent of LiCl concentration in the film. Initially, the salt release is controlled by the nearly constant-rate absorption of water. The salt release, at long times, lags behind the swelling-controlled water uptake, indicating that the salt release is not completely controlled by the water sorption and that diffusion in the swollen polymer matrix contributes significantly to the long term elution of LiCl. Independent thermal analysis experiments suggest the formation of a metal salt—poly(ethylene—vinyl alcohol) complex.     

Viscometric behaviour of polymer blends based on poly (vinylidene fluoride)

The viscosity behaviour of dilute dimethylformamide solutions of poly(vinylidene fluoride)-poly (methyl methacrylate) and poly(vinylidene fluoride)-polystyrene has been studied at 25°C. The polymer concentration ranges are such that neither phase separation nor microgel formation occurs, although we are very close to theta conditions. The intrinsic viscosity and viscosity interaction parameter of the ternary mixtures have been calculated. The estimation of the compatibility of the above polymer pairs has been studied based on: a) specific viscosities; b) viscosity interaction parameters, according to Krigbaum and Wall formalism, and c) viscosity interaction parameters of a system formed by a dilute probe polymer in the presence of a matrix polymer and a small molecule solvent.     

Phase separation kinetics of polyelectrolyte solutions

The kinetics of phase separation of aqueous solutions of sodium-poly(styrene sulfonate) (NaPSS) containing barium chloride (BaCl(2)) is studied by static and dynamic light scattering. We report a novel mechanism of phase separation, where an enrichment of polymer aggregates of well-defined size occurs in the very early stage of nucleation, which is then followed by a growth process in the formation of the new phase. In the latter stage, the polymer aggregates formed in the early stage act as the templating nuclei. Even in the homogeneous phase at higher temperatures above the upper critical phase boundary, polymer aggregates are present in agreement with previously reported results. Upon rapidly cooling the system below the phase boundary, the number concentration of the aggregates increases first by maintaining their size to be relatively monodisperse, before the growth process takes over at later times. The size and fractal dimension of aggregates in the homogeneous phase and the early nucleation stage of phase separation and the dependence of nucleation time and growth rate on quench depth and salt concentration are determined. The hydrodynamic radius (R(H)) of the unaggregated chains is of the order of 1-10 nm depending on the molecular weight of NaPSS, while R(H) of aggregates is of the order of 100 nm independent of the molecular weight of NaPSS. Unaggregated chains follow good solution behavior with a fractal dimension of 5/3 while the fractal dimension of aggregates is larger than 3.5 suggesting the branched nature of aggregates. Nucleation time is sensitive to quench depth and salt concentration. Increasing a quench depth or increasing BaCl(2) concentration shortens the nucleation time. After the nucleation time, during the growth period, the size of aggregates grows linearly with time, with growth rate being higher for deeper quench depths and higher BaCl(2) concentrations. The mechanism of phase separation of aqueous solutions of NaPSS and BaCl(2) is seen to proceed by utilizing the already-existing aggregates to nucleate the new phase, in marked contrast to hitherto known results on phase separation in uncharged polymer systems.