Development of a gel-free molecular sieve based on self-assembled nano-arrays

#The present invention is directed to a self-assembled nano-array molecular sieve for the separation of molecules.

#This invention relates in general to self-assembled nanometer-scale arrays used as molecular sieves in the separation of molecules by differential transport through the array, and in particular to self-assembled carbon nanotube arrays used as electrophoretic sieves for DNA sequencing and separation of biological molecules comprising a self-assembled carbon nanotube array arranged on a substrate and an electromagnetic field generator for applying a potential across the array sieve producing a characteristic mobility in the molecules.

#Electrophoresis is the predominant technique for separating DNA fragments obtained from restriction maps of complete genomes (millions of base pairs long) and for large-scale sequencing projects, like the Human Genome Initiative. Electrophoresis has also become an essential tool for clinical chemistry applications. In conventional electrophoretic sieves, electrophoretic separation occurs by differential transport of polyelectrolytes, such as, DNA molecules and proteins, through a medium or device in the presence of an electric field. The medium acts as a sieve, producing a size-dependent mobility in the molecules. In slab gel electrophoresis, the sieving medium is provided by a slab gel of agarose or polyacrylamide polymers, which contain nanometer-size pores. For example, an agarose gel is made by placing agarose into solution with a suitable solvent and the pore size depends on the concentration of agarose in solution according to the equation:

#These and other features and advantages of the present invention will be better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings wherein:

#FIG. 1 is a schematic view of an embodiment of a nano-array sieve according to the invention.

#FIG. 2 is a schematic view of an embodiment of a nano-array sieve in operation according to the invention.

#The present invention is directed to a molecular sieve instrument comprising a non-gel self-assembled nano-scale periodic array sieve for discriminating molecules based on a characteristic mobility. As shown in FIG. 1, the molecular sieve 10 comprises a substrate 12 having proximal 14 and distal ends 16 and an array 18 of self-assembled nano-features comprising an ordered repeating pattern of nano-features 20 arranged on the substrate 12 of the molecular sieve 10. In operation, an electric field 22 is applied across the length of the array 18 from the proximal 14 to the distal 16 end such that molecules 24 introduced onto the array 18 surface move through the array 18 to a detector (not shown) in a time dependent on a characteristic of the molecule 24, such as, for example size, charge, chemical inertness, etc.

#Although in the embodiment shown in FIG. 1, the self-assembled array 18 of nano-features comprises periodic rows of uniform nano-features 20 attached to the substrate 12, any other arrangement of nano-features 20 suitable to function as a molecular sieve 10 can be utilized such as, for example, staggered arrays of uniform nano-features or uniform rows of nano-features having alternating sizes. In another alternative embodiment the spacing of the nano-features 20 is varied to produce a gradient gel for which sieving properties vary spatially. Additionally, while FIG. 1 only shows a molecular sieve 10 deposited on a flat substrate surface 12, any geometry of substrate 12 suitable for use as a molecular sieve 10 could be utilized, such as, for example, a curved, corrugated or tubular substrate. It will be understood that the design of the molecular sieve 10 according to the present invention is necessarily driven by the molecules 24 to be separated as the sieving ability of the molecular sieve 10 is sensitively dependent on both the geometrical size, shape and spacing of the nano-features 20 as well as the size and properties of the molecules 24. The molecular sieve 10 itself has two characteristic sizes: (1) the nano-feature size and (2) the nano-feature spacing, or pore size. Using an iterative process, the nano-features 20 can be optimized to separate molecules 24 of a specified size or other physical property, then other parameters can be selected to optimize operation, such as, for example, electric field 22 strength, detector and the mechanical properties of the sieve body. Accordingly, although the self-assembled molecular sieve 10 of the embodiment shown in FIG. 1 comprises a uniform array 18 of uniform cylindrical nano-features 20, it should be understood that any shape, size or spacing of nano-features 20, as described above, can be utilized in the molecular sieve 10 of the current invention such that the pore size and sieving properties of the array 10 are suitable for use as a separation device for the molecules 24 of interest.

#For example, a sieve must possess specific properties to separate double strand DNA (dsDNA) and single strand DNA (ssDNA) molecules. The DNA molecule generally appears in its dsDNA configuration. The dsDNA molecule can span a wide variety of sizes, ranging from a few kilo-base-pairs (kbp) to many Mbp, such as, for example, chromosomes. To provide sequencing capabilities, on the other hand, the molecular sieve 10 must be able to process ssDNA molecules which are produced by Maxam-Gilbert or Sanger sequencing reactions. In contrast to the dsDNA molecule, the ssDNA molecule used for sequencing is typically no more than 1,000 bases long. Accordingly, the design of a molecular sieve 10 intended to separate either form of DNA requires that the conformational behavior of ssDNA and dsDNA under electrophoresis be known. This behavior, in turn, strongly relies on the physical properties of the array 18 and sieve 10. For example, if the gaps or pores between the nano-features 20 in the arrays 18 are smaller than 30 nm, pulsed electrophoresis utilizing a pulsed electric field 22, which requires the bending of DNA, may not be feasible because of the minimum bending diameter of dsDNA (Db˜30 nm). Also, the value of the electric field 22 E compared to the electric field E0 required to balance the thermal motion of Kuhn segment will influence the stretching configuration of the molecules 24. The relaxation time, or Rouse time TR, associated with the molecules may also impose a limit on the design dimensions of a molecular sieve 10 for sequencing. Table 1, below, compares the physical properties of the ssDNA and dsDNA molecules.