Patent Abstract:
A nanoscale-sized pore positioned between two reservoirs may sequence biomolecules by detecting changes in the emitted light due to a change in charge of portions of the biomolecules as they pass through the pore such as affect an emission frequency of a quantum structure proximate to the pore opening. The nanopores may be fabricated using local droplet etching whose randomness is accommodated by lowering the droplet density to permit isolation of nanopores in tiles that may be adhered to an underlying supporting substrate having an aligned opening. The nanopore-tiles may be integrated with commonly applied glass chips and may be employed in microfluidic circuitry.

Full Description:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     CROSS REFERENCE TO RELATED APPLICATION 
     BACKGROUND OF THE INVENTION 
       [0001]    The present invention relates to a system for the direct sequencing of polymers such as DNA and RNA by passing the polymer through a nanoscale pore and measuring a light signal modulated by the polymer. 
         [0002]    Genetic information is encoded in a molecule of deoxyribonucleic acid (DNA) as a sequence of nucleotides: guanine, adenine, thymine, and cytosine. Discovering the sequence of these nucleotides in DNA and other similar molecules is a foundational technology in biological studies. 
         [0003]    One promising method of sequencing is “nanopore sequencing” in which a single strand of DNA, forming half of the DNA helix, is passed through a nanoscale opening in a membrane between two reservoirs. This nanopore opening may, for example, be a protein channel held in a lipid bilayer. An electrical potential or other gradient (i.e. molar concentrations. thermal, etc.) may be applied across the reservoirs to produce an ion flow between the reservoirs pulling the strand of DNA through the nanopore. As the strand passes through the nanopore, it modulates the ion current through the nanopore as a function of the size of the nucleotide obstructing the nanopore. This alteration in the ion current may then be analyzed to determine the nucleotide sequence. An example system of nanopore sequencing is described in PCT patent WO/2008102120 entitled: “Lipid Bilayer Sensor System”, and in European patent 2695949 entitled: “Nucleic Acid-based Nano Pores or Transmembrane Channels and their Uses”, both hereby incorporated by reference. 
         [0004]    The electrical signals produced by changes in ion current through a nanopore with different nucleotides are very small in amplitude and accordingly long sampling times are required to distinguish the signals from noise, resulting in a slowing down of the sequencing process. The ability to obtain required sampling times may not be available because of the high speed of motion of the DNA strand through the nanopore. 
       SUMMARY OF THE INVENTION 
       [0005]    The present invention provides a sequencing apparatus using an optically active nanopore. The nanopore includes a semiconductor nanoscale structure near the pore opening exhibiting quantum confinement effects that are affected by the electrical field of the long chain molecule passing through. Field-induced changes in the band gap of the nanoscale structure, as different portions of the long chain molecule pass through the nanopore, cause the emission of light at different frequencies such as may be mapped to different structures of the long chain molecule. 
         [0006]    More specifically, one embodiment the invention provides an apparatus for a measurement of biomolecules using a separator with a nanopore providing a passage through the separator, the nanopore incorporating a nanoscale semiconductor element proximate to the passage that is adapted to emit light with a frequency dependent on a charge of a portion of biomolecules passing through the nanopore. A reservoir system holds a fluid on opposite sides of the separator to provide a flow of biomolecules through the nanopore from one side of the separator to the other and a spectrometer receives emitted light from the nanoscale semiconductor element to measure frequency of that light as biomolecules flow through the nanopore. An electronic computer communicates with the spectrometer and executes a program to relate light frequency measured by the spectrometer to structure of the portion of the biomolecules thereby providing a sequencing of biomolecule structures as the biomolecules passes through the nanopore. 
         [0007]    It is thus a feature of at least one embodiment of the invention to provide an improved (high-speed) sensing structure for sequencing biomolecules making use of local interaction between the biomolecule and a light-emitting quantum confinement structure. 
         [0008]    The nanoscale semiconductor element may be a ring concentric with the nanopore and bounded by different materials on nanoscale dimensions to provide a structure exhibiting quantum confinement effects. 
         [0009]    It is thus a feature of at least one embodiment of the invention to provide a “quantum ring” sensor that may maximize sensitivity of the quantum structure to the electrical field of a material within the ring. 
         [0010]    The different materials bounding the ring may also be semiconducting materials potentially increasing the sensitivity of the detection mechanism. 
         [0011]    It is thus a feature of at least one embodiment of the invention to provide a quantum structure that may be readily fabricated using integrated circuit techniques on semiconductor materials. 
         [0012]    The nanoscale semiconductor element and different materials may be group III/V semiconductors but can also be group II/VI. 
         [0013]    It is thus a feature of at least one embodiment of the invention to provide materials that may produce a light output more easily communicated from the nanopore to a measuring instrument. 
         [0014]    The semiconductor may be selected from the group consisting of: gallium arsenide, aluminum gallium arsenide, and indium arsenide. 
         [0015]    It is thus a feature of at least one embodiment of the invention to provide a nanoscale semiconducting structure employing well-characterized materials. 
         [0016]    The apparatus may further include a light source for providing stimulating energy to the nanoscale semiconductor element to promote an emission of light from the nanoscale semiconductor element. 
         [0017]    It is thus a feature of at least one embodiment of the invention to promote light emission by providing a source of stimulating energy that may be tailored to the hand gap of the nanoscale semiconductor element. 
         [0018]    The separator may be a solid material substantially unbroken outside of the nanopore over an area contacting fluid of the reservoir structure. 
         [0019]    It is thus a feature of at least one embodiment of the invention to eliminate the need for fragile lipid bilayers normally used as separators between reservoirs in favor of a substantially continuous and more robust solid-state separator. 
         [0020]    The separator may be a membrane holding the nanopore and adhered to a substrate of different material, the substrate having an aperture aligned with the nanopore. 
         [0021]    It is thus a feature of at least one embodiment of the invention to permit fabrication of separators with nanopores using techniques, such as a local droplet etching, which have limited working depth by attaching a thin membrane etched using these techniques to a thicker substrate while aligning an opening in the two. 
         [0022]    The nanopore may be substantially circular or may be non-circular in cross-section, either sized to control an orientation of the biomolecule as it passes through the nanopore, 
         [0023]    It is thus a feature of at least one embodiment of the invention to promote the sequencing of biomolecules by mechanically constraining the flow of biomolecules through the nanopore. 
         [0024]    The invention also provides a method of manufacturing separators of the type that can isolate reservoirs of liquid across at least one optically active nanopore. In the method, a matrix material is fabricated on a sacrificial layer supported by a first substrate and subjected to local droplet etching in which metal droplets erode nanoscale holes through the matrix material to the sacrificial layer. A second substrate is then prepared with a plurality of apertures larger than the nanoscale holes, wherein the second substrate is thicker than the matrix material. The matrix material is then removed from the sacrificial layer and adhered to the second substrate so that at least one nanopore aligns with at least one aperture. The adhered matrix material and second substrate are then divided into multiple separator elements each including a continuous passage through a nanoscale hole and aperture. 
         [0025]    It is thus a feature of at least one embodiment of the invention to provide a robust separator that can eliminate the need for bilayer lipid membranes and yet still provide nanoscale holes. 
         [0026]    The method may include separating the matrix material into a plurality of tiles after the local droplet etching and independently attaching the tiles to the second supporting substrate. 
         [0027]    It is thus a feature of at least one embodiment of the invention to accommodate the random distribution of nanoscale holes obtained by techniques such as local droplet etching. 
         [0028]    The matrix material may be adhered to the second substrate by van-der-Waals forces. 
         [0029]    It is thus a feature of at least one embodiment of the invention to provide a method of attaching the matrix material to a supporting structure that permits a period of adjustment before permanently affixing the matrix material to the supporting structure. 
         [0030]    The method may include the step of coating the nanoscale hole with a semiconductor material different from a material of the walls of the nanoscale hole. 
         [0031]    It is thus a feature of at least one embodiment of the invention to permit the construction of a quantum confinement element proximate to a nanoscale opening for use in sequencing biomolecules and other similar applications. 
         [0032]    These particular features and advantages may apply to only some embodiments falling within the claims and thus do not define the scope of the invention. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0033]      FIG. 1  is a block diagram of a sequencing apparatus employing an optically active nanopore in exaggerated scale as positioned in a separator between reservoirs of fluid; 
           [0034]      FIG. 2  is a detailed cross-section of the nanopore of  FIG. 1  during fabrication, showing a semiconductor matrix material supported on a sacrificial layer attached to a construction substrate immediately after being etched by local droplet etching; 
           [0035]      FIG. 3  is an expanded cross-section of the nanopore during use showing additional layers applied to the opening of the nanopore to create a quantum structure having conduction and valence bands providing a bandgap that varies with electrical interaction between the quantum structure and a proximal molecule being analyzed, and further showing in an inset, a spectrum of emitted light such as varies with such electrical interaction; 
           [0036]      FIG. 4  is a simplified flowchart showing the formation of a separator of  FIG. 1  by the attachment of nanopores in a matrix material to the supporting substrate; and 
           [0037]      FIG. 5  is a top plan depiction of an elliptical and circular nanopore such as may be created with the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0038]    Referring now to  FIG. 1 , an apparatus  10  for characterizing molecules passing through a nanopore may comprise a generally rigid planar separator  12  extending along a plane  15  and having an opening  14  passing through the separator  12  generally perpendicular to the plane  15 . 
         [0039]    A reservoir structure having first and second reservoirs  16   a  and  16   b  may be constructed on either side of the separator  12  about the opening  14  to be separated from each other by the separator  12  and communicating only through the opening  14 . These reservoirs  16   a  and  16   b  may be filled with a conductive fluid  20  such as a buffer solution, for example, KCl solution, as held by capillary attraction or a fluidic channel. Reservoir  16   a  may have an introduced source of biomolecules  22  (for example, single DNA strands or double strand DNA helices and the necessary proteins and enzymes to separate the helix into strands) suspended therein. 
         [0040]    Each of the reservoirs  16   a  and  16   b  may hold electrodes  23  (for example, silver/silver chloride electrodes) communicating between with the liquid of the reservoir structure and a voltage source  24  together to provide an electrical voltage across the opening  14  tending to produce an ionic flow from reservoir  16   a  to reservoir  16   b.  This flow may draw the biomolecules  22  along with it causing individual biomolecules  22  to thread through the opening  14 . As monomers  25  of the biomolecules  22  pass through the opening  14 , different electrical charges associated with each monomer  25  may influence a quantum structure  27  proximate to the opening  14 . 
         [0041]    In particular, during the flow of the biomolecules  22 , the quantum structure  27  may be excited with a light beam  28 , for example, from ultraviolet light source  30  focusing a beam of ultraviolet light on the quantum structure  27 , for example, through microscope objective  32 . In response to this excitation, the quantum structure  27  will emit light  34  which may pass upward through the microscope objective  32  along the path of the light beam  28  and then be separated by a beam splitter  36  from the light beam  28  and then directed toward a spectrometer  38 . As will be discussed below, the frequency of this emitted light  34  will be affected by electrical charges associated with different monomers  25  of the biomolecules  22  as they pass through the nanopore  26  generating a unique fingerprint for each monomer  25  related to the light frequency. 
         [0042]    The spectrometer  38  receiving the frequency modulated emitted light  34  provides a frequency output  40  indicating a center frequency of the emitted light  34 . This frequency output  40  is then received by an electronic computer  42  for analysis. As is generally understood in the art, the electronic computer  42  may include one or more processing elements  44  communicating with a memory  46  holding a stored program executable on the processing elements  44  to analyze the frequency output  40 . The computer  42  may also control the light source  30  both to turn it on and off and optionally to adjust its intensity and/or frequency to improve the signal-to-noise ratio of the measured emitted light  34 . 
         [0043]    The computer  42  executing a stored program will in turn provide sequence information  48 , for example, presented on an electronic display  50  or the like, providing a time sequence  52  of measurements indicating the sequence of monomers  25  in the biomolecules  22  as a function of time thereby sequencing the biomolecules  22 . In the case of a DNA biomolecule  22 , the monomers  25  will be guanine, adenine, thymine and cytosine whose different electrical fields provide different frequency modulation of the emitted light  34 . 
         [0044]    In one embodiment, the separator  12  holding the nanopore  26  through which the biomolecules  22  pass, may be a laminated structure, for example, including a tile  54  of a semiconductor matrix material  55  holding the actual nanopore  26  supported on a support substrate  56  having a larger aperture  60  having diameters from 1 to 10 micrometers. 
         [0045]    The tile  54  of semiconductor matrix material  55  may be of relatively small area, for example, several tens of microns on each side of a square perimeter and may have a thickness between 20 and 400 nanometers. Tile  54  will be relatively flexible and in some manufacturing processes may include multiple nanopores  26  and for this reason is supported on the upper face of the larger support substrate  56 , for example, the latter constructed of borosilicate glass quartz of much greater thickness (measured perpendicularly to plane  15 ), for example, in a range from 0.1 millimeters to 1.2 millimeters or more. 
         [0046]    As noted, the nanopore  26  will be aligned with the larger aperture  60  in the support substrate  56 , the latter such as may be fabricated using a laser according the technique described in U.S. Pat. No. 8,092,739 “Retro-Percussive Technique For Creating Nanoscale Holes” and U.S. Pat. No. 8,623,496 “Laser Drilling Technique for Creating Nanoscale Holes” assigned to the assignee of the present invention and hereby incorporated by reference. In this technique, an ultraviolet absorbent liquid is confined to the back-side of a quartz substrate to absorb energy when pulsed by an excimer laser passing through the substrate. 
         [0047]    Referring now to  FIG. 2 , the nanopore  26  may be formed in the matrix material  55  by means of local droplet etching (LDE) of the type described in “Local droplet etching of nanoholes and rings on GaAs and Al GaAs surfaces”, A. Steinmann, Ch. Heyn, T. Köppenl, T. Kipp and W. Hansen Appl. Phys. Lett. 93, 123108 (2008) and “Scaling of the structural characteristics of nanoholes created by local droplet etching”, Ch. Heyn, S. Schnüll and W. Hansen, J. Appl. Phys. 115, 024309 (2014), hereby incorporated by reference, 
         [0048]    In this process, the matrix material  55 , for example, an aluminum gallium arsenide semiconductor, may be fabricated on a sacrificial layer  62 , for example, of silicon dioxide or aluminum arsenide, this sacrificial layer  62  in turn supported by a much thicker and substantially rigid fabrication substrate  64 , for example, a silicon wafer. Sacrificial layer  62  is selectively removable by a solvent such as hydrofluoric acid so as to permit release of the matrix material  55  from the fabrication substrate  64  without damage to the matrix material  55 , 
         [0049]    While the matrix material  55  is held on the fabrication substrate  64  by the sacrificial layer  62 , metal droplets  66  are then formed on its surface through a nucleation process described in the above-cited references. In one embodiment these metal droplets  66  may be gallium or indium. During a post-growth thermal annealing step, the droplets  66  create nanopits  67  into the matrix material  55 . While the inventors do not wish to be bound by a particular theory, it is believed that the central process for this etching is a diffusion of arsenic from the matrix material  55  into the metal droplet and subsequent droplet material removal. 
         [0050]    The resulting nanopit  67  extends through the matrix material  55  into the sacrificial layer  62  and becomes a nanopore  26  when the sacrificial layer  62  is removed. When the droplet  66  is gallium on an aluminum gallium arsenide matrix material  55 , the inner walls  68  of the nanopore  26  will be predominantly gallium arsenide in contrast to the aluminum gallium arsenide of the matrix material  55 . When the droplet  66  is indium, the material of the inner walls  68  will be indium arsenide. This technique may also be used with a gallium arsenide matrix material  55  in which ease the material of the inner wall  68  may be gallium arsenide with a different material property than the matrix material  55  caused by higher amounts of gallium, or indium arsenide, 
         [0051]    The average diameter of the nanopore  26  will be less than 1000 nanometers. 
         [0052]    Referring now to  FIG. 3 , this inner wall  68  may be covered with a coating  70  of aluminum gallium arsenide (in the case of an aluminum gallium arsenide matrix material  55 ) to sandwich the inner wall  68  between two dissimilar semiconducting materials of aluminum gallium arsenide. The thickness of the inner wall  68  measured in the plane  15  may be less than 10 nanometers to provide quantum structure  27  having quantum confinement effects as will be discussed and as are caused by the confining effects of the coating  70  and material of the matrix material  55 , In addition a capping layer  72 , for example, gallium arsenide may be placed over the coating  70 . Generally gallium arsenide is compatible with DNA and thus a suitable capping material for capping layer  72 ; however, other materials may be used to provide a nonreactive outer surface with different biomolecules  22 . 
         [0053]    The semiconductor materials of matrix material  55 , inner walls  68 , and coating  70  including capping layer  72  create a set of adjacent conduction bands  74  and valence bands  76  of different energies that produce a quantum confinement in the inner walls  68  as bounded by inner walls  68  and capping layer  72  as is necessary for optical emissions. This bounding of the inner wall  68  provides quantum structure  27 . 
         [0054]    Generally the semiconductor materials may be selected from group MN or group however other material such as strained silicon germanium may be used. The group MN materials may be selected from gallium arsenide, aluminum gallium arsenide, and indium arsenide. 
         [0055]    A bandgap  78  between the conduction bands  74  and valence band  76  of the inner walls  68  define a bandgap energy which determines the frequency of light emitted from the inner wall  68  when electrons drop between the conduction bands  74  and valence band  76 . The value of this bandgap energy separating the valence and conduction bands  76  and  74  of the inner wall  68  will change slightly in response to the electric field  80  associated with monomers  25  of the biomolecule  22  proximate to the inner walls  68 . The result will be a change in the frequency  82  of emitted light  34  depending on the monomer  25 . 
         [0056]    This change in emitted light frequency  82  is detected by the spectrometer  38  shown in  FIG. 1  and a measurement of center frequency transmitted to the computer  42 . A program running on the computer  42  may employ a predetermined a set of frequency zones  84  which are empirically determined and then used in mapping frequency of emitted light  34  to different zones  84  associated with different monomers  25 . Accordingly, movement of the frequency  82  of the emitted light  34  among the zones  84  provides a characterization of the monomers  25  and hence a sequencing of the biomolecule  22  as it passes through the nanopore  26 . 
         [0057]    Referring now to  FIG. 4 , fabrication of the separators  12  is complicated by the largely random process of nucleation used in local droplet etching. Accordingly the present invention, in one embodiment, controls the local droplet etching to reduce the density of holes produced to be, for example, an average one nanopore  26  for every tile  54  (for example, one nanopore  26  for every 10 to 100 micrometer square). As will be discussed below, multiple nanopores  26  may be accommodated on an individual tile  54 . Separation of the matrix material  55  into the tiles for may be accomplished by defining on the matrix material  55  a grid using optical lithography to provide an etch mask for a dry etch along boundaries between the tiles  54 . Finally a selective wet edge step may be carried out so that the tiles  54  released float off the carrier surface. 
         [0058]    The support substrate  56  is then prepared with the regular spacing of larger apertures  60  and the separated tiles  54  placed on the support substrate  56  so that the nanopores  26  align with larger apertures  60  with the tiles  54 . Adjustment of the tiles  54  on the support substrate  56  may be accomplished while the tiles  54  are wet and may float on a liquid layer held by capillary force between the tile  54  and the support substrate  56 . Once properly located, the tile  54  is firmly attached to the support substrate  56  by van-der-Waals forces, which come into play when the liquid layer between the tile  54  and the support substrate  56  evaporates. 
         [0059]    Alternatively dry tiles  54  may be placed directly on the support substrate  56  with suction enabled micro-pipettes  63  aligning the dry tiles  54  with the larger apertures  60  per arrows  61 . 
         [0060]    The support substrate  56  may then be cut into the final dimension of the separators  12  as described above, each separator  12  holding one tile  54  and providing one functioning nanopore  26 . It will be appreciated that each tile  54  may have multiple nanopores  26  and that the alignment of a single nanopore  26  with a single larger aperture  60  may serve to limit the number of channels passing through each separator  12 . 
         [0061]    In one embodiment, the WE etched matrix material  55  may be bonded directly to the support substrate  56  without being separated into tiles  54  with the expectation that the randomly located nanopores  26  will randomly align with some of the larger apertures  60 . These locations of alignment may then be determined, for example, by observation using photoluminescence measurements or the like and used to guide the division of the matrix material  55  and support substrate  56  into the separators  12 . 
         [0062]    It is contemplated that in some embodiments, separators  12  with multiple nanopores  26  aligned with larger aperture  60  may be provided to be used for parallel sequencing operations where multiple biomolecules  22  are sequenced in parallel using separate light collectors and separate spectrographic analysis. 
         [0063]    Referring now to  FIG. 5 , it will be appreciated that the nanopore  26  may have a circular opening  94  (seen along the viewing axis normal to the plane  15 ) or by taking advantage of anisotropic characteristics of the semiconductor matrix material  55 , for example, crystal axis directions, which may have an ellipsoidal opening  96 . This latter shape may be desirable for sequencing of bio-molecules that are not circular in cross-section. For example, this opening  96  may be sized to roughly conform to a lateral extension of the monomers  25  in the plane  15  thereby provoking a rotation of the biomolecule  22  as it passes through the nanopore  26  such as may assist in controlling the progression of the biomolecule  22  through the nanopore  26  Or to select among different biomolecules, Alternatively this orientation may assist in providing more standardized effects of the electrostatic field of the monomers  25  On the surrounding quantum structure  27  formed from inner wall  68 . 
         [0064]    In this document, “different materials” refers to materials having different band energies and includes materials with different doping concentrations such as may be suitable for creating a quantum confinement structure. 
         [0065]    Certain terminology is used herein for purposes of reference only, and thus is not intended to be limiting. For example, terms such as “upper”, “lower”, “above”, and “below” refer to directions in the drawings to which reference is made. Terms such as “front”, “back”, “rear”, “bottom” and “side”, describe the orientation of portions of the component within a consistent but arbitrary frame of reference, which is made clear by reference to the text and the associated drawings describing the component under discussion. Such terminology may include the words specifically mentioned above, derivatives thereof, and Words of similar import. Similarly, the terms “first”, “second” and other such numerical terms referring to structures do not imply a sequence or order unless clearly indicated by the context. 
         [0066]    When introducing elements or features of the present disclosure and the exemplary embodiments, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of such elements or features. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements or features other than those specifically noted. It is further to be understood that the method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed. 
         [0067]    References to a “processor” or “processor unit” should generally be understood to refer broadly to general-purpose computer processing elements for executing stored programs (software) comprised of sequences of arithmetic and logical operations stored in the general-purpose memory. The term “circuit” as used herein should be considered to broadly include both analog and digital circuitry together with associated firmware. The term “program” generally refers to a sequence of operations executed by a processor or circuit. References to memory, unless otherwise specified, can combinations of different memory structures including solid-state and electromechanical memories and may describe a distributed system of main memory and multiple cache layers. 
         [0068]    It is specifically intended that the present invention not be limited to the embodiments and illustrations contained herein and the claims should be understood to include modified forms of those embodiments including portions of the embodiments and combinations of elements of different embodiments as come within the scope of the following claims. All of the publications described herein, including patents and non-patent publications, are hereby incorporated herein by reference in their entireties.

Technology Classification (CPC): 1