The present invention relates generally to nanopore and micropore devices and methods for their manufacture and use. More particularly the present invention relates to nanopore and micropore devices used for detecting, and characterizing biomolecules, as well as sequencing nucleic acids, and new methods for fabrication and application thereof.
Nanopores and micropores are structures that are useful and implemented for a wide range of applications. Nanopores are holes with diameters in the range of 100 nm to 1 nm in a membrane or solid media. Micropores refer to holes with diameters ranging from 100 nm to 100 μm. For nanopores, many applications have been contemplated including rapid detection and characterization of biological agents, and DNA sequencing. Micropores are already widely used as a mechanism for separating cells.
For the applications of DNA sequencing, two prior-art methods have been proposed using nanopores. U.S. Pat. No. 5,795,782, issued to Church et al. discloses a method of reading DNA sequence by detecting the ionic current variations as a single-stranded DNA molecule moving through the nanopore under a bias voltage. Another method for DNA sequencing using nanopores was discussed in U.S. Pat. No. 6,537,755, issued to Drmanac. Drmanac proposes to use nanopores to detect the DNA hybridization probes (oligonucleotides) on a DNA molecule and recover the DNA sequence information using the method of Sequencing-By-Hybridization (SBH).
Other applications using nanopores have been discussed widely in literature, including real-time monitoring of cell activities and detection of biological agents in biodefense. For many of these important applications, it is highly desirable to develop nanopore devices using solid-state materials. Here solid-state materials are broadly defined including high-density materials such as silicon oxides and nitrides, and polymeric materials such as hard plastics, as they are naturally insulating or can be insulated by adding a surface layer or making the surface insulating, e.g. by oxidization.
A well-known technique for producing nanoscale holes in solid-state materials uses high-energy particles to create damage tracks. The tracks become nanoscale holes when etched. Because a freestanding nanometer-thick film is mechanically unstable, the nanopores produced by this technique often have length on the order of a micrometer or more and are typically limited by the thickness of the starting film. For example, such long nanopores (also called nano-capillaries) were used by Bohn et al., in US Patent Publication No. 2003/0136679 and Kuo et al., in “Hybrid three-dimensional nanofluidic/microfluidic devices using molecular gates”, Sensors and Actuators A, vol. 102 (October 2002): 223-233. Such long nanopores are not suitable for many applications such as DNA sequencing as currently discussed. Recent efforts in this field have been focused on developing new techniques for fabricating short nanopores having a pore length that is less than 20 nm.
U.S. Pat. No. 6,464,842, issued to Golovchenko et al. proposed a film-thinning technique. In the disclosed method, a shallow cavity is first created on one side of a membrane; the membrane is then thinned down from the opposite side slowly using a low-energy ion beam. Utilizing an active feedback loop to control the application of the low-energy ion beam, one can stop the thinning process as soon as a pore is opened at the cavity. This technique has been further developed into a nanoscale “sculpting” approach, [see Li et al., “Ion-beam sculpting at nanometer length scales”, Nature, vol. 412 (July 2001): 166-169]. The main drawback of this approach is that highly specialized and expensive ion-beam instrumentation is needed.
In U.S. Pat. No. 6,503,409, Fleming proposes a method of making a nanopore (or nano-aperture) using silicon technology. In Fleming's approach, a nanopore is formed at the crossing point of two nanoslits. The nanoslits are fabricated by etching away a thin (2-5 nm) layer of sacrificial material. Because the pore size is controlled by the thickness of the sacrificial layer, the channel connecting to the pore is also of 2-5 nm in thickness. This feature is highly undesirable since the DNA molecules will be less likely to access the pore due to the large penalty in free energy (known as the entropic barrier) they have to overcome to reach the pore.
To take advantage of the silicon technology while avoiding the limit of the Fleming approach, Storm et al. [Storm et al., “Fabrication of solid-state nanopores with single-nanometer precision”, Nature Materials, vol. 2 (August 2003): 537-540.] proposed a different method. Storm et al. used standard electron-beam lithography to create a 100 nm etch mask on the surface of a silicon membrane. When this mask is exposed to KOH solutions, an inverse pyramid feature is developed due to the anisotropy of the etching rate along different crystalline axes. Storm et al. showed that one can simply control the etching time so that a pore of 20 nm is opened on the opposite side of the etch mask. After oxidization, the silicon around the pore opening is turned into silicon oxide (SiO2). In the next step the SiO2 material around the pore is fluidized using a high-energy electron beam, such that the surface tension of the fluidized SiO2 pulls the material into the empty space thereby reducing the pore size. This technique has been shown to have great reliability in producing nanopores with diameters ranging from 1-20 nm. The pores can also be completely closed off using the same technique.
There are at least two major limitations in all of the above discussed techniques related to nanopore fabrication. The first is that all of the above techniques only produce individual pores. A single nanopore in a membrane will have very low throughput in molecule sensing operations. Accordingly, it is highly desirable to have an array of nanopores on the same membrane. However, a device using a membrane with many parallel pores will not be useful, since one cannot determine which pore a DNA is moving through. Second, all of the above techniques produce nanopores having pore length on the order of 10 nm or more.
In order to make the nanopores useful for practical applications, one needs to create a high-throughput device such as an array of nanopores wherein each pore in the array is independently electrically addressable. It would be desirable to provide a device and method that yields a linear array of nanopores or two-dimensional array of nanopores. It also would be particularly desirable to provide such linear and two-dimensional nanopore arrays that are electrically addressable. It would further be desirable to provide a new nanopore that can be formed using synthetic material and a method for using and making such a nanopore. It also would be desirable to provide a nanopore or an array of nanopores each with a length of less than 10 nm. It would be highly desirable to have simplified processes for fabricating nanopores and related fluidic systems at low cost.
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