We have established the nanofabrication technique for constructing nanopillars with high aspect ratio (100-500 nm diameter and 500-5000 nm tall) inside a microchannel on a quartz chip. The size of pillars and the spacing between pillars are designed as a DNA sieving matrix for optimal analysis of large DNA fragments over a few kilobase pairs (kbp). A chip with nanopillar channel and simple cross injector was developed based on the optimal design and applied to the separation of DNA fragments (1 - 38 kbp) and large DNA fragments (λ DNA, 48.5 kbp; T4 DNA, 165.6 kbp) that are difficult to separate on conventional gel electrophoresis and capillary electrophoresis without a pulsed-field technique. DNA fragments ranging from 1 to 38 kbp were separated as clear bands, and furthermore, the mixture of λ DNA and T4 DNA was successfully separated by a 380-μm-long nanopillar channel within only 10 s even under a direct current (dc) electric field. Theoretical plate number N of the channel (380-1450 μm long) was 1000-3000 (0.7 × 106-2.1 × 106 plates/m). A single DNA molecule observation during electrophoresis in a nanopillar channel revealed that the optimal nanopillars induced T4 DNA to form a narrow U-shaped conformation during electrophoresis whereas λ DNA kept a rather spherical conformation. We demonstrated that, even under a dc electric field, the optimal nanopillar dimensions depend on a gyration radius of DNA molecule that made it possible to separate large DNA fragments in a short time.
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