Separation of Long DNA Molecules by Quartz Nanopillar Chips under a Direct Current Electric Field

Abstract

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 (lambda 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 lambda DNA and T4 DNA was successfully separated by a 380-microm-long nanopillar channel within only 10 s even under a direct current (dc) electric field. Theoretical plate number N of the channel (380-1450 microm long) was 1000-3000 (0.7 x 10(6)-2.1 x 10(6) 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 lambda 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.

Nanofluidic structures for single biomolecule fluorescent detection

“…[36][37][38] A single DNA strand may be visualized as it interacts with pillars in a microfluidic slit, [39][40][41] or as it escapes from a deep well into a thin slit, and it is hoped that the knowledge gained from such studies will lead to faster methods of lab-on-chip DNA sorting. [42][43][44][45][46][47][48] Finally, sequence information may even be obtained from stretched molecules through restriction site mapping or motif mapping using hybridized probes. 19,20,49 In addition to stretching via hydrodynamic forces, channels, planar slits, and pillar arrays, fabricated into glass or silicon wafers can also be used to study the mechanical dynamics of a DNA molecule by employing entropic forces that arise due to confinement.…”

Collision of a DNA Polymer with a Small Obstacle

“…The first experimental work to separate O[10-100 kbp] DNA by length using hooking collisions was by Doyle et al 3 Followup work was performed in slightly more dense obstacle courses at higher fields 21 and in extremely dense courses, 22 though as the obstacle density increases, the separation mode switches from unhooking to pore sieving characteristic of gels. Motivated by these experiments, macroscale separation models were proposed to explain the dependence of the average mobility and dispersivity in an obstacle array on the DNA length.…”

Filling kinetics of liquids in nanochannels as narrow as 27 nm by capillary force

“…For bionanotechnology, studying single molecules, and physicochemical phenomena of liquids in the nanometer scale nanofluidic devices provide a novel tool [1][2][3][4][5][6]. Downsizing of fluidic systems is attractive for fundamental studies as well as biosensing [7][8][9].…”

“…Downsizing of fluidic systems is attractive for fundamental studies as well as biosensing [7][8][9]. For example, nanometersized entropic traps were used to separate long, genomic DNA [2,3]. Nanochannels combined with near-field microscopy could be used to locate genes [10].…”