Nanopores, methods for using same, methods for making same and methods for characterizing biomolecules using same

Abstract

Featured are devices and systems embodying one or more solid-state nanopores useable for sensing and/or characterizing single macromolecules as well as sequencing DNA or RNA and/or determining RNA secondary structures. In once solid state nanopore of the present invention the width and/or length of the nanopore is defined or established by sharp edges of cleaved crystals that are maintained in fixed relation during the formation of the insulating member including the nanopore. In another aspect of the present invention, there is featured a linear or 2-D electrically addressable array of nanopores, where the nanopores are located at points of intersections between grooves formed in an upper surface of the insulating member and a groove formed in a lower surface of the insulating member.

1 . A device for characterizing biomolecules, comprising: 
 an insulating member, the insulating member including a through aperture extending between opposing surfaces; and    wherein a width of the through aperture is established by a plurality of crystals that are arranged in fixed relation with respect to each other while forming the insulating member.    
   
   
       2 . The device of  claim 1 , wherein edges of the plurality of crystals cross each other at a predetermined angles and are maintained in this arrangement while forming the insulating member.  
   
   
       3 . The device of  claim 2 , wherein the edges form or define an area that is small enough that the molecules making up the insulating member cannot enter into this area while forming the insulating member.  
   
   
       4 . The device of  claim 2 , wherein molecules of insulating material are oriented with respect to the edges thereby forcing the insulating material to form a contour around the crossing point thereby defining the through aperture in the insulating member.  
   
   
       5 . A method for characterizing a biomolecule comprising the steps of: 
 providing an insulating member, the insulating member including a through aperture extending between opposing surfaces and wherein a width of the through aperture is established by a plurality of crystals that are arranged in fixed relation with respect to each other while forming the insulating member;    locating the insulating member between ionic reservoirs at least one of which includes the biomolecule to be characterized; and    passing the biomolecule through the through aperture of the provided insulating member.    
   
   
       6 . The method of clam  5 , further comprising the step of: 
 detecting an ionic current and changes in ionic current as the biomolecule is passed through the through aperture; and    characterizing the biomolecule based on the detected ionic current and changes thereto.    
   
   
       7 . A method for determining RNA secondary structures comprising the steps of: 
 providing an insulating member, the insulating member including a through aperture extending between opposing surfaces and wherein a width of the through aperture is established by a plurality of crystals that are arranged in fixed relation with respect to each other while forming the insulating member;    locating the insulating member between ionic reservoirs at least one of which includes the biomolecule to be characterized;    operably coupling coupling one end of the RNA to an optical tweezer; and    measuring a force at said one end as the RNA molecule is pulled through the through aperture.    
   
   
       8 . An electrically-addressable nanopore array comprising: 
 an insulating material member;    wherein the insulating material layer is configured and arranged so as to have a plurality or more of grooves extending lengthwise in a first direction in a first surface thereof;    wherein the insulating material member is configured and arranged so as to have a groove extending lengthwise in a second direction in a second surface thereof, the first and second surfaces being opposed to each other;    wherein the second direction is at an angle with respect to the first direction; and    wherein the grooves are formed in each of the first and second surfaces so that at an intersection of each off the grooves in the first surface and the groove in the second surface there is formed an opening that comprises a nanopore.    
   
   
       9 . The electrically-addressable nanopore array of  claim 8 , wherein the insulating material members is formed from a first layer and a second layer, where a surface of the first layer is the first surface of the insulating material member, where the first layer includes the plurality of grooves formed in the insulating member first surface, where a surface of the second layer is the second surface of the insulating material member and where the second layer includes the groove formed in the insulating member second surface.  
   
   
       10 . The electrically-addressable nanopore array of  claim 9 , wherein the first and second layers are one of bounded or secured to each so as to form the insulating material member.  
   
   
       11 . The electrically-addressable nanopore array of  claim 8 , wherein the insulating material member is configured and arranged so as to have a plurality of grooves extending lengthwise in a second direction in the second surface thereof.  
   
   
       12 . The electrically-addressable nanopore array of  claim 11 , wherein the insulating material layer is configured and arranged so as to have a plurality of sets of a plurality or more of grooves extending lengthwise in a first direction in a first surface thereof, where the grooves in each set are not connected to grooves in another set.  
   
   
       13 . The electrically-addressable nanopore array of  claim 12 , wherein the grooves are formed in each of the first and second surfaces so that at an intersection of each off the grooves in the first surface of each set of grooves and one of the plurality of grooves in the second surface there is formed an opening that comprises a nanopore.  
   
   
       14 . A method for characterizing a biomolecule comprising the steps of: 
 providing an electrically-addressable nanopore array including an insulating material member, wherein the insulating material member is configured and arranged so as to have a plurality or more of grooves extending lengthwise in a first direction in a first surface thereof and a groove extending lengthwise in a second direction in a second surface thereof, the second direction is at an angle with respect to the first direction, and wherein the grooves are formed in each of the first and second surfaces so that at an intersection of each off the grooves in the first surface and the groove in the second surface there is formed an opening that comprises a nanopore;    locating the electrically-addressable nanopore array between ionic reservoirs at least one of which includes the biomolecule to be characterized; and    passing the biomolecule through one of the nanopores of the provided electrically-addressable nanopore array.    
   
   
       15 . The method of clam  14 , further comprising the step of: 
 detecting an ionic current and changes in ionic current as the biomolecule is passed through the one the nanopores; and    characterizing the biomolecule based on the detected ionic current and changes thereto.    
   
   
       16 . A method for determining RNA secondary structures comprising the steps of: 
 providing an electrically-addressable nanopore array including an insulating material member, wherein the insulating material member is configured and arranged so as to have a plurality or more of grooves extending lengthwise in a first direction in a first surface thereof and a groove extending lengthwise in a second direction in a second surface thereof, the second direction is at an angle with respect to the first direction, and wherein the grooves are formed in each of the first and second surfaces so that at an intersection of each off the grooves in the first surface and the groove in the second surface there is formed an opening that comprises a nanopore;    locating the electrically-addressable nanopore array between ionic reservoirs at least one of which includes the biomolecule to be characterized;    operably coupling coupling one end of the RNA to an optical tweezer; and    measuring a force at said one end as said the RNA molecule is pulled through one of the nanopores.    
   
   
       17 . A nanopore comprising: 
 an insulating member having opposed surfaces; and    a through aperture extending between said opposing surfaces.    
   
   
       18 . The nanopore of  claim 17 , wherein the width of said through aperture is less than about 10 nm.  
   
   
       19 . A nanopore array comprising a plurality of nanopores, wherein each nanopore comprises: 
 an insulating member having opposed surface; and    a through aperture extending between said opposing surfaces.    
   
   
       20 . The nanopore array of  claim 19 , wherein said through aperture has a width that is less than about 10 nm.  
   
   
       21 . The nanopore array of  claim 19 , wherein each of said nanopores is electronically addressable.  
   
   
       22 . A method for preparing a nanopore comprising an insulating member having opposed surfaces and a through aperture extending between said surfaces, the method comprising the steps of: 
 forming an aperture having a width that is established by fixing a plurality of crystals fixed in position to one another;    forming an insulating member by casting an insulating material over said plurality of crystals fixed in position relative to one another, thereby forming the insulating material with said aperture extending through opposing surfaces of said insulating material; and    removing said plurality of crystals,    to thereby prepare a nanopore comprising an insulating member having opposed surfaces and a through aperture extending between said surfaces.    
   
   
       23 . A nanopore comprising an insulating member having opposed surfaces and a through aperture extending between said surfaces, said nanopore having been prepared by a method comprising the steps of: 
 forming an aperture having a width that is established by fixing a plurality of crystals fixed in position to one another;    forming an insulating member by casting an insulating material over said plurality of crystals fixed in position relative to one another, thereby forming the insulating material with said aperture extending through opposing surfaces of said insulating material; and    removing said plurality of crystals,    to thereby prepare a nanopore comprising an insulating member having opposed surfaces and a through aperture extending between said surfaces.    
   
   
       24 . A method for preparing a nanopore array comprising an insulating member having opposed first and seconds surfaces and one or more through apertures extending between said surfaces, the method comprising the steps of: 
 forming one or more grooves in the first surface that extend lengthwise in a first direction;    forming one or more grooves in the second surface that extend lengthwise in a first direction, the second direction being at an angle with respect to the first direction;    removing material at intersections of the one or more grooves in the first surface and the one or more grooves in the second surface;    to thereby prepare a nanopore comprising an insulating member having opposed surfaces and a one or more through aperture extending between said surfaces.    
   
   
       25 . The device of  claim 2 , wherein the edges of the crystals are spaced from each other a distance (Se) and the crystal distance spacing is set so as to satisfy (Se/Tm)≦0.5 where Tm is a thickness of a molecule of the material making up the insulating member.  
   
   
       26 . The device of  claim 2 , wherein the edges of the crystals are spaced from each other a distance (Se) and the crystal distance spacing is set so as to be in the range of from about 1 Å to about 10 Å.  
   
   
       27 . The device of  claim 1 , wherein the through aperture has a length less than or equal 20 Å.  
   
   
       28 . The device of  claim 1 , wherein the through aperture has a length (d) that satisfies the relation 2 Å≦d≦10 Å.  
   
   
       29 . The method of  claim 5 , wherein the through aperture of the provided insulating member has a length less than or equal 20 Å.  
   
   
       30 . The method of  claim 1 , wherein the through aperture of the provided insulating member has a length (d) that satisfies the relation 2 Å≦d≦10 Å.  
   
   
       31 . The method of  claim 5 , wherein the edges of the crystals are spaced from each other a distance (Se) and the crystal distance spacing is set so as to satisfy (Se/Tm)≦0.5 where Tm is a thickness of a molecule of the material making up the insulating member.  
   
   
       32 . The method of  claim 5 , wherein the edges of the crystals are spaced from each other a distance (Se) and the crystal distance spacing is set so as to be in the range of from about 1 Å to about 10 Å.  
   
   
       33 . The method of  claim 7 , wherein the through aperture of the provided insulating member has a length less than or equal 20 Å.  
   
   
       34 . The method of  claim 7 , wherein the through aperture of the provided insulating member has a length (d) that satisfies the relation 2 Å≦d≦10 Å.  
   
   
       35 . The method of  claim 7 , wherein the edges of the crystals are spaced from each other a distance (Se) and the crystal distance spacing is set so as to satisfy (Se/Tm)≦0.5 where Tm is a thickness of a molecule of the material making up the insulating member.  
   
   
       36 . The method of  claim 7 , wherein the edges of the crystals are spaced from each other a distance (Se) and the crystal distance spacing is set so as to be in the range of from about 1 Å to about 10 Å.  
   
   
       37 . A method for preparing a two-dimensional nanopore array comprising an insulating member having opposed surfaces and a plurality of through aperture extending between said surfaces, the method comprising the steps of: 
 forming each of the plurality of apertures having a width that is established by fixing a plurality of crystals fixed in position to one another for each aperture;    forming an insulating member by casting an insulating material over each of said plurality of crystals fixed in position relative to one another for each aperture, thereby forming the insulating material with said each aperture extending through opposing surfaces of said insulating material; and    removing said plurality of crystals for each aperture,    to thereby prepare a two-dimensional nanopore array comprising an insulating member having opposed surfaces and a plurality of through apertures extending between said surfaces.    
   
   
       38 . A method for preparing a nanopore array comprising the steps of: 
 providing a plurality of nanopore assemblies, each of the plurality of nanopore assemblies including an insulating member having opposed surfaces and a through aperture extending between said surfaces,    assembling the plurality of nanopore assemblies so the through apertures form at least a one-dimensional array of through apertures; and    wherein said providing a plurality of nanopore assemblies includes performing the steps of for each nanopore assembly: 
 forming an aperture having a width that is established by fixing a plurality of crystals fixed in position to one another,  
 forming an insulating member by casting an insulating material over said plurality of crystals fixed in position relative to one another, thereby forming the insulating material with said aperture extending through opposing surfaces of said insulating material, and  
 removing said plurality of crystals,  
 to thereby prepare a nanopore comprising an insulating member having opposed surfaces and a through aperture extending between said surfaces.  
   
   
   
       39 . The method of  claim 38 , wherein said assembling the plurality of nanopore assemblies includes assembling the plurality of nanopore assemblies so the through apertures form a two-dimensional array of through apertures.  
   
   
       40 . The method of any of claims  38 - 39 , wherein the through aperture of each of the plurality of provided nanopore assemblies has a length less than or equal 20 Å.  
   
   
       41 . The method of any of claims  38 - 39 , wherein the through aperture of each of the plurality of provided nanopore assemblies has a length (d) that satisfies the relation  
       2 Å≦d≦10 Å.  
   
   
       42 . The method of any of claims  38 - 39 , wherein said forming an aperture includes spacing edges of the crystals from each other a distance (Se), where the distance is set so as to satisfy the relation (Se/Tm)≦0.5 where Tm is a thickness of a molecule of the material making up the insulating member.  
   
   
       43 . The method of any of claims  38 - 39 , wherein said forming an aperture includes spacing edges of the crystals from each other a distance (Se), where the distance is set so as to be in the range of from about 1 Å to about 10 Å.  
   
   
       44 . A method for sequencing DNA or RNA comprising the steps of: 
 providing an insulating member, the insulating member including a through aperture extending between opposing surfaces and wherein a width of the through aperture is established by a plurality of crystals that are arranged in fixed relation with respect to each other while forming the insulating member;    coating at least inner surfaces of the through aperture with a PNA including one of the A, T, G or C bases that characterize DNA/ RNA;    locating the insulating member between ionic reservoirs at least one of which includes the DNA/RNA to be sequenced;    operably coupling coupling one end of the DNA/RNA to an optical tweezer; measuring a force as a function of time at said one end as said DNA/RNA is pulled through the through aperture; and    correlating the measured force as a function of time to one of A, T, G, or C bases,    to thereby prepare sequencing the DNA or RNA.    
   
   
       45 . A method for sequencing DNA or RNA comprising the steps of: 
 providing a plurality of nanopore assemblies, each of the plurality of nanopore assemblies including an insulating member having opposed surfaces and a through aperture extending between said surfaces,    assembling the plurality of nanopore assemblies so the through apertures form at least a one-dimensional array of through apertures;    coating at least inner surfaces of the through aperture of each of the plurality of nanopore assemblies with a PNA including one of the A, T, G or C bases that characterize DNA/RNA;    locating the insulating member of each nanopore assembly so an ionic reservoir which includes the DNA/RNA to be sequenced is disposed on a side of at least one of the plurality of nanopore assemblies;    operably coupling coupling one end of the DNA/RNA for the through aperture of said at least one nanopore assembly to an optical tweezer;    measuring a force as a function of time at said one end as said DNA/RNA is pulled through the through aperture of said at least one nanopore assembly;    correlating the measured force as a function of time to one of A, T, G, or C bases, to thereby sequencing the DNA or RNA; and    wherein said providing a plurality of nanopore assemblies includes performing the steps of for each nanopore assembly: 
 forming an aperture having a width that is established by fixing a plurality of crystals fixed in position to one another,  
 forming an insulating member by casting an insulating material over said plurality of crystals fixed in position relative to one another, thereby forming the insulating material with said aperture extending through opposing surfaces of said insulating material,  
 removing said plurality of crystals, and  
 coating at least inner surfaces of the through aperture with a PNA including one of the A, T, G or C bases that characterize DNA/RNA;  
   
   
   
       46 . The device of  claim 1 , further comprising a coating of a PNA including one of the A, T, G or C bases that characterize DNA/RNA; wherein the coating is applied so as to coat at least inner surfaces of the through aperture.  
   
   
       47 . The electrically-addressable nanopore array of  claim 8 , further comprising a coating of a PNA including one of the A, T, G or C bases that characterize DNA/RNA; wherein the coating is applied so as to coat at least inner surfaces of the opening formed at the intersections of each of the grooves in the first and second surfaces.  
   
   
       48 . The nanopore of  claim 17 , further comprising a coating of a PNA including one of the A, T, G or C bases that characterize DNA/RNA; wherein the coating is applied so as to coat at least inner surfaces of the through aperture.  
   
   
       49 . The nanopore array of  claim 19 , further comprising a coating of a PNA including one of the A, T, G or C bases that characterize DNA/RNA; wherein the coating is applied so as to coat at least inner surfaces of the through aperture for each of the plurality of nanopores.  
   
   
       50 . A method for sequencing DNA or RNA comprising the steps of: 
 providing any one of the device of  claim 46 , the electrically-addressable nanopore array of  claim 47 , the nanopore of  claim 48  and the nanopore array of  claim 49;     locating said any one of the device, the electrically-addressable nanopore array, the nanopore and the nanopore array so as to be between ionic reservoirs at least one of which includes the DNA/RNA to be sequenced;    operably coupling coupling one end of the DNA/RNA to an optical tweezer;    measuring a force as a function of time at said one end as said DNA/RNA is pulled through the through aperture; and    correlating the measured force as a function of time to one of A, T, G, or C bases,    to thereby prepare sequencing the DNA or RNA.    
   
   
       51 . The method for preparing a nanopore according to  claim 22 , further comprising the step of coating at least inner surfaces of the through aperture with a PNA including one of the A, T, G or C bases that characterize DNA/RNA;  
   
   
       52 . The method for preparing a nanopore array according to  claim 24 , further comprising the step of coating at least inner surfaces of the one or more through apertures with a PNA including one of the A, T, G or C bases that characterize DNA/RNA;