Brett A. Buchholz

Critical factors for high-performance physically adsorbed (dynamic) polymeric wall coatings for capillary electrophoresis of DNA

Physically adsorbed (dynamic) polymeric wall coatings for microchannel electrophoresis have distinct advantages over covalently linked coatings. In order to determine the critical factors that control the formation of dynamic wall coatings, we have created a set of model polymers and copolymers based on N,N‐dimethylacrylamide (DMA) and N,N‐diethylacrylamide (DEA), and studied their adsorption behavior from aqueous solution as well as their performance for microchannel electrophoresis of DNA. This study is revealing in terms of the polymer properties that help create an “ideal” wall coating. Our measurements indicate that the chemical nature of the coating polymer strongly impacts its electroosmotic flow (EOF) suppression capabilities. Additionally, we find that a critical polymer chain length is required for polymers of this type to perform effectively as microchannel wall coatings. The effective mobilities of double‐stranded (dsDNA) fragments within dynamically coated capillaries were determined in order to correlate polymer hydrophobicity with separation performance. Even for dsDNA, which is not expected to be a strongly adsorbing analyte, wall coating hydrophobicity has a deleterious influence on separation performance.

Poly-N-hydroxyethylacrylamide (polyDuramide ): A novel, hydrophilic, self-coating polymer matrix for DNA sequencing by capillary electrophoresis

A replaceable polymer matrix, based on the novel monomer N‐hydroxyethylacrylamide (HEA), has been synthesized for application in DNA separation by microchannel electrophoresis. The monomer was found by micellar electrokinetic chromatography analysis of monomer partitioning between water and 1‐octanol to be more hydrophilic than acrylamide and N,N‐dimethylacrylamide. Polymers were synthesized by free radical polymerization in aqueous solution. The weight‐average molar mass of purified polymer was characterized by tandem gel permeation chromatography‐multiangle laser light scattering. The steady‐shear rheological behavior of the novel DNA sequencing matrix was also characterized, and it was found that the viscosity of the novel matrix decreases by more than 2 orders of magnitude as the shear rate is increased from 0.1 to 1000 s–1. Moreover, in the shear‐thinning region, the rate of change of matrix viscosity with shear rate increases with increasing polymer concentration. Poly‐N‐hydroxyethylacrylamide (PHEA) exhibits good capillary‐coating ability, via adsorption from aqueous solution, efficiently suppressing electroosmotic flow (EOF) in a manner comparable to that of poly‐N,N‐dimethylacrylamide. Under DNA sequencing conditions, adsorptive PHEA coatings proved to be stable and to maintain negligible EOF for over 600 h of electrophoresis. Resolution of DNA sequencing fragments, particularly fragments > 500 bases, in PHEA matrices generally improves with increasing polymer concentration and decreasing electric field strength. When PHEA is used both as a separation matrix and as a dynamic coating in bare silica capillaries, the matrix can resolve over 620 bases of contiguous DNA sequence within 3 h. These results demonstrate the good potential of PHEA matrices for high‐throughput DNA analysis by microchannel electrophoresis.

Impact of polymer hydrophobicity on the properties and performance of DNA sequencing matrices for capillary electrophoresis.

To elucidate the impact of matrix chemical and physical properties on DNA sequencing separations by capillary electrophoresis (CE), we have synthesized, characterized and tested a controlled set of different polymer formulations for this application. Homopolymers of acrylamide and N,N‐dimethylacrylamide (DMA) and copolymers of DMA and N,N‐diethylacrylamide (DEA) were synthesized by free radical polymerization and purified. Polymer molar mass distributions were characterized by tandem gel permeation chromatography ‐ laser light scattering. Polymers with different chemical compositions and similar molar mass distributions were selected and employed at the same concentration so that the variables of comparison between them were hydrophobicity and average coil size in aqueous solution. We find that the low‐shear viscosities of 7% w/v polymer solutions decrease by orders of magnitude with increasing polymer hydrophobicity, while hydrophilic polymers exhibit more pronounced reductions in viscosity with increased shear. The performance of the different matrices for DNA sequencing was compared with the same sample under identical CE conditions. The longest read length was produced with linear polyacrylamide (LPA) while linear poly‐N,N‐dimethylacrylamide (PDMA) gave ˜ 100 fewer readable bases. Read lengths with DMA/DEA copolymers were lower, and decreased with increasing DEA content. This study highlights the importance of polymer hydrophilicity for high‐performance DNA sequencing matrices, through the formation of robust, highly‐entangled polymer networks and the minimization of hydrophobic interactions between polymers and fluorescently‐labeled DNA molecules. However, the results also show that more hydrophobic matrices offer much lower viscosities, enabling easier microchannel loading at low applied pressures.

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.

Comb-like copolymers as self-coating, low-viscosity and high-resolution matrices for DNA sequencing

Comb‐like copolymers with a polyacrylamide backbone and poly(N,N‐dimethylacrylamide) grafts were prepared, as a way to combine the superior sieving properties of polyacrylamide with the self‐coating properties of polydimethylacrylamide. These matrices function well in the absence of a capillary coating, and achieve separation performances for single‐stranded DNA that are comparable to those of state‐of‐the‐art long‐chain linear polyacrylamide. Structural parameters such as the grafting density and the polymer molecular mass were varied, and good performance appears to be achieved with a relatively large range of parameters. Surprisingly, excellent separation is achieved even with matrices that have a viscosity as low as 200 mPa/s. A discussion of the physics underlying this behavior is provided.

The use of light scattering for precise characterization of polymers for DNA sequencing by capillary electrophoresis.

The ability of a polymer matrix to separate DNA by capillary electrophoresis (CE) is strongly dependent upon polymer physical properties. In particular, recent results have shown that DNA sequencing performance is very sensitive to both the average molar mass and the average coil radius of the separation matrix polymers, which are affected by both polymer structure and polymer‐solvent affinity. Large polymers with high average molar mass provide the best DNA sequencing separations for CE, but are also the most challenging to characterize with accuracy. The methods most commonly used for the characterization of water‐soluble polymers with application in microchannel electrophoresis have been gel permeation chromatography (GPC) and intrinsic viscosity measurements, but the limitations and potential inaccuracies of these approaches, particularly for large or novel polymers and copolymers, press the need for a more universally accurate method of polymer molar mass profiling for advanced DNA separation matrices. Here, we show that multi‐angle laser light scattering (MALLS) measurements, carried out either alone or in tandem with prior on‐line sample fractionation by GPC, can provide accurate molar mass and coil radius information for polymer samples that are useful for DNA sequencing by CE. Wider employment of MALLS for characterization of novel polymers designed as DNA separation matrices for microchannel electrophoresis should enable more rapid optimization of matrix properties and formulation, and assist in the development of novel classes of polymer matrices.

DNA sequencing with hydrophilic and hydrophobic polymers at elevated column temperatures

Read length in DNA sequencing by capillary electrophoresis at elevated temperatures is shown to be greatly affected by the extent of hydrophobicity of the polymer separation matrix. At column temperatures of up to 80°C, hydrophilic linear polyacrylamide (LPA) provides superior read length and separation speed compared to poly(N,N‐dimethylacrylamide) (PDMA) and a 70:30 copolymer of N,N‐dimethylacrylamide and N,N‐diethylacrylamide (PDEA30). DNA‐polymer and polymer intramolecular interactions are presumed to be a major cause of band broadening and the subsequent loss of separation efficiency with the more hydrophobic polymers at higher column temperatures. With LPA, these interactions were reduced, and a read length of 1000 bases at an optimum temperature of 70°–75°C was achieved in less than 59 min. By comparison, PDMA produced a read length of roughly 800 bases at 50°C, which was close to the read length attained in LPA at the same temperature; however, the migration time was approximately 20% longer, mainly because of the higher polymer concentration required. At 60°C, the maximum read length was 850 bases for PDMA, while at higher temperatures, read lengths for this polymer were substantially lower. With the copolymer DEA30, read length was 650 bases at the optimum temperature of 50°C. Molecular masses of these polymers were determined by tandem gel permeation chromatography‐multiangle laser light scattering method (GPC‐MALLS). The results indicate that for long read, rapid DNA sequencing and analysis, hydrophilic polymers such as LPA provide the best overall performance.

A Novel, Hydrophilic, Adsorptive Polymeric Coating for Electrophoretic Separation of DNA and Proteins in Silica Microchannels

A novel, replaceable polymer matrix based on a new N-substituted acrylamide monomer Duramide™ has been synthesized for application in DNA separations by microchannel electrophoresis. The monomer was found to be more hydrophilic than both acrylamide and N, N-dimethylacrylamide (DMA). Polymers were synthesized by free radical polymerization in aqueous solution and characterized by tandem gel permeation chromatography — multi-angle laser light scattering. PolyDuramide™ exhibits good capillary-coating ability via adsorption from aqueous solution, efficiently suppressing electroosmotic flow (EOF) in a manner comparable to that of poly-N,N-dimethylacrylamide (PDMA). Under DNA sequencing conditions, adsorptive polyDuramide™ coatings proved to be stable and to maintain negligible EOF for over 600 hours of electrophoresis. Resolution of DNA sequencing fragments, particularly fragments > 500 bases, in the novel matrix generally improves with increasing polymer concentration and decreasing electric field strength. Using polyDuramide both as a separation matrix and as a dynamic coating in bare fused silica capillaries, the matrix can resolve over 620 bases of contiguous DNA sequence within about 3 hours. Within a glass chip, the same results could likely be achieved in ∼30 minutes. These results demonstrate the potential of the novel matrix for use in DNA separation, as well as proteins, by microchannel electrophoresis.