(141b) Nanoporous Polymer Membrane for DNA Preconcentration Enables Sensitive Chip Electrophoresis

Conventional chip electrophoresis using a double-T injection design enables high peak efficiency due to the narrow injection plug, but detection sensitivity is often limited because this format offers relatively little sample stacking, and much of the sample is wasted simply filling the injection area. Numerous other approaches have been demonstrated for on-line preconcentration of DNA samples, including isotachophoresis, micropatterned electrodes, and sequence-specific ?capture matrices?. Large concentration factors are possible, but each technique presents certain drawbacks in terms of simplicity of device fabrication or operation.

We report on the use of thin (~30 micron) photopatterned polymer membranes for on-line preconcentration of single- or double-stranded DNA samples prior to electrophoretic analysis. Shaped UV laser light is used to quickly (~10 seconds) polymerize a narrow, highly crosslinked polyacrylamide plug. By applying an electric field across this membrane, DNA from a dilute sample can be concentrated into a narrow zone (<100 micron wide) at the outside edge of the membrane. The field at the membrane can then be reversed, allowing the narrow plug to be cleanly injected into a separation channel filled with a sieving polymer for analysis. Concentration factors >100 are possible, increasing the sensitivity of analysis for dilute samples. We have fabricated both neutral membranes (purely size-based exclusion) as well as anionic membranes (size and charge exclusion), and characterized the rate of preconcentration as well as the efficiency of injection from both types of membrane, for several sizes of DNA, ranging from a 20 base ssDNA oligonucleotide to >14 kbp dsDNA. We have also investigated the effects of concentration polarization on device performance for the charged membrane. Results for sensitivity as well as separation efficiency are compared to those for the Agilent Bioanalyzer, a widespread and successful platform for chip electrophoresis.

Advantages of the membrane preconcentration approach include the simplicity of device fabrication and operation, and the generic (non-sequence specific) nature of DNA capture, which is useful for complex or poorly characterized samples where a specific capture sequence is not present. The membrane preconcentration approach is well suited to simple single-level etch glass chips, with no need for patterned electrodes, integrated heaters, valves, or other elements requiring more complex chip fabrication.