Microfabricated Porous Membrane Structure for Sample Concentration and Electrophoretic Analysis

Detection capabilities in microchip electrophoresis for biological applications can be enhanced by integration of on-chip sample concentration methods in the analysis. A microfabricated injection valve incorporating a porous membrane structure has been developed that enables electrokinetic concentration of DNA samples using homogeneous buffer conditions followed by injection into a channel for electrophoretic analysis. The porous membrane was incorporated in the microchannel manifold by having two channels separated from each other by 3 - 12 µm and connected by a thin porous silicate layer. This design allows the passage of current to establish an electrical connection between the separated channels, but prevents large molecules, e.g. DNA, from traversing the membrane. Concentrated DNA can be injected into the separation channel and electrophoretically analyzed. Experiments exhibit a non-linear increase in concentration with time, and DNA fragments can be concentrated up to 2 orders of magnitude.

Figure 1 shows the design of a porous membrane microchip used for DNA preconcentration and electrophoretic analysis. The schematic of the porous membrane structure is presented in Figure 2. The injection tee and side channel are separated from each other by a distance of 3 to 12 µm, depending on the mask used and the etch time. A silicate bonding process was employed to join the substrate and cover plate glass surfaces and the resulting thin polysilicate layer also provides the porous membrane between the analyte and side channels. CCD images in Figure 3 illustrate double-stranded (ds) DNA concentration in the tee region of the channel adjacent to the membrane followed by injection of the preconcentrated plug into the separation channel. During sample loading DNA molecules accumulate in the tee-region due to their hindered transport through the membrane, and the concentration, observed by monitoring the fluorescence intensity, increased with time (Figure 3b and 3c). Reconfiguration of the applied potential distribution for the separation mode results in an injection of the concentrated DNA plug into the separation channel (Figure 3d). Typical electropherograms of ds DNA fragments, concentrated on-chip for different periods of time, are shown in Figure 4. Fluorescence response increases with concentration time non-linearly and a DNA sample can be concentrated up to 2 orders of magnitude prior to electrophoretic analysis without a significant loss of the separation performance.Microfabricated porous membrane structures could allow increased throughput and enhance integrated DNA analysis on-chip.

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