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

ABSTRACT: 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.

SUBMITTER: Sun M

PROVIDER: S-EPMC4346347 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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

Sun Mingyun M Lin Jennifer S JS Barron Annelise E AE

Electrophoresis 20111018 22

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-mol ...[more]

PMID: 22009451

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Project description:Alginate hydrogels are widely used as delivery vehicles due to their ability to encapsulate and release a wide range of cargos in a gentle and biocompatible manner. The release of encapsulated therapeutic cargos can be promoted or stunted by adjusting the hydrogel physiochemical properties. However, the release from such systems is often skewed towards burst-release or lengthy retention. To address this, we hypothesized that the overall magnitude of burst release could be adjusted by combining microgels with distinct properties and release behavior. Microgel suspensions were generated using a process we have termed on-chip polymer blending to yield composite suspensions of a range of microgel formulations. In this manner, we studied how alginate percentage and degradation relate to the release of lentivectors. Whereas changes in alginate percentage had a minimal impact on lentivector release, microgel degradation led to a 3-fold increase, and near complete release, over 10 days. Furthermore, by controlling the amount of degradable alginate present within microgels the relative rate of release can be adjusted. A degradable formulation of microgels was used to deliver vascular endothelial growth factor (VEGF)-encoding lentivectors in the chick chorioallantoic membrane (CAM) assay and yielded a proangiogenic response in comparison to the same lentivectors delivered in suspension. The utility of blended microgel suspensions may provide an especially appealing platform for the delivery of lentivectors or similarly sized therapeutics.Statement of significanceGenetic therapeutics hold considerable potential for the treatment of diseases and disorders including ischemic cardiovascular diseases. To realize this potential, genetic vectors must be precisely and efficiently delivered to targeted regions of the body. However, conventional methods of delivery do not provide sufficient spatial and temporal control. Here, we demonstrate how alginate microgels provide a basis for developing systems for controlled genetic vector release. We adjust the physiochemical properties of alginate for quicker or slower release, and we demonstrate how combining distinct formulations of microgels can tune the release of the overall composite microgel suspension. These composite suspensions are generated using a straightforward and powerful application of droplet microfluidics which allows for the real-time generation of a composite suspension.

Dataset Information

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

Publications

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

Similar Datasets

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