Patent Publication Number: US-2022236250-A1

Title: Dual pore sensors

Description:
BACKGROUND 
     Field 
     Embodiments herein relate to flow cells to be used with solid-state nanopore sensors and methods of manufacturing thereof. 
     Description of the Related Art 
     Solid-state nanopore sensors have emerged as a low-cost, easily transportable, and rapid processing biopolymer, e.g., DNA or RNA, sequencing technology. Solid-state nanopore sequencing of a biopolymer strand typically incudes translocating a biopolymer strand through one or more nanoscale sized openings each having a diameter between about 0.1 nm and about 100 nm, i.e., a nanopore. In a single pore sensor, a nanopore is disposed through a membrane layer which separates two conductive fluid reservoirs. The biopolymer strand to be sequenced, e.g., a characteristically negatively charged DNA or RNA strand, is introduced into one of the two conductive fluid reservoirs and is then drawn through the nanopore by providing an electric potential therebetween. As the biopolymer strand travels through the nanopore the different monomer units thereof, e.g., protein bases of a DNA or RNA strand, occlude different percentages of the nanopore thus changing the ionic current flow therethrough. The resulting current signal pattern can be used to determine the sequence of monomer units in the biopolymer strand, such as the sequence of proteins in a DNA or RNA strand. Generally, single pore sensors lack a mechanism for slowing the rate of translocation of the biopolymer strand through the nanopore while still providing sufficient electrical potential between the two reservoirs to optimize the signal to noise ratio in the resulting current signal pattern. 
     Beneficially, dual pore sensors provide a mechanism for controlling the rate of translocation of a biopolymer strand by co-capturing the biopolymer strand in the two nanopores thereof. A typical dual pore sensor features two fluid reservoirs separated by a wall, a common fluid chamber, and a membrane separating the common fluid chamber from each of the fluid reservoirs, the membrane layer having the two nanopores disposed therethrough. A biopolymer strand to be sequenced travels from the first fluid reservoir to the common chamber and from the common chamber to the second fluid reservoir through a second nanopore. Desirably the two nanopores are positioned close enough to one another to allow for co-capture of the biopolymer strand. When the biopolymer strand is co-captured by both of the nanopores, competing electric potentials are applied across each of the nanopores to create a “tug-of-war” where the opposite ends of the biopolymer strand are pulled in opposite directions of travel. Beneficially, the difference between the competing electric potentials can be adjusted to control the rate of translocation of the biopolymer strand through the nanopores and thus the resolution of the electrical signal current signal pattern or patterns resulting therefrom. 
     Often, dual nanopore sensors are formed using two substrates. Typically, the first substrate is formed of an amorphous non-monocrystalline material, such as glass, which is patterned to form the first and second fluid reservoirs having the wall disposed therebetween. The second substrate is formed of monocrystalline silicon and a multi-layer stack comprising the membrane layer is formed on a surface thereof. The membrane layer of the second substrate is then anodically bonded to the patterned surface of the first substrate, the silicon substrate is removed from the multi-layer stack, and an opening is etched into the multilayer stack to form the common chamber. The nanopores are then formed through respective portions of the membrane layer disposed on either side of the wall using a focused ion beam (FIB) drilling process. 
     Unfortunately, the manufacturing methods described above are generally incompatible with the high volume manufacturing, quality, repeatability, and cost requirements needed to move dual pore sensors out of the R&amp;D lab and into the public market. Further, the manufacturing methods described above generally limit the minimum spacing between the two nanopores to about 550 nm which thus limits the ability of dual pore sensors formed therefrom to sequence relativity shorter biopolymer strands. 
     Accordingly, there is a need in the art for improved methods of forming dual pore sensors and improved dual pore sensors formed therefrom. 
     SUMMARY 
     Embodiments of the present disclosure provide solid state dual pore sensors which may be used for biopolymer sequencing, and methods of manufacturing the same. 
     In one embodiment, a method of forming a dual pore sensor includes providing a pattern in a surface of a substrate. Generally, the pattern features two fluid reservoirs separated by a divider wall. The method further includes depositing a layer of sacrificial material into the two fluid reservoirs, depositing a membrane layer, patterning two nanopores through the membrane layer, removing the sacrificial material from the two fluid reservoirs, and patterning one or more fluid ports and a common chamber. 
     In another embodiment, a dual pore sensor features a substrate having a patterned surface comprising two recessed regions spaced apart by a divider wall and a membrane layer disposed on the patterned surface. The membrane layer, the divider wall, and one or more surfaces of each of the two recessed regions collectively define a first fluid reservoir and a second fluid reservoir. A first nanopore is disposed through a portion of the membrane layer disposed over the first fluid reservoir and a second nanopore is disposed through a portion of the membrane layer disposed over the second fluid reservoir. Herein, opposing surfaces of the divider wall are sloped to each form an angle of less than 90° with a respective reservoir facing surface of the membrane layer. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       So that the manner in which the above recited features of the present disclosure can be understood in detail, a more particular description of the disclosure, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this disclosure and are therefore not to be considered limiting of its scope, for the disclosure may admit to other equally effective embodiments. 
         FIG. 1A  is a close up sectional view schematically illustrating a portion of a dual pore sensor formed using one or a combination of the embodiments described herein. 
         FIG. 1B  schematically illustrates an anisotropically etched surface of a silicon substrate. 
         FIG. 2  is a flow diagram setting forth a method of forming a dual pore sensor, according to one or more embodiments. 
         FIGS. 3A-3K  schematically illustrate various aspects of the results of the method set forth in  FIG. 2 . 
         FIG. 3L  schematically illustrates an aspect of the results of an alternative embodiment of the method set forth in  FIG. 2 . 
         FIGS. 4A-4B  schematically illustrate various aspects of the results of an alternative embodiment of the method set forth in  FIG. 2 . 
         FIG. 5  is a plan view of a substrate having a plurality of dual pore sensors formed thereon, according to one embodiment. 
     
    
    
     DETAILED DESCRIPTION 
     Embodiments of the present disclosure provide solid state dual pore sensors which may be used for biopolymer sequencing, and methods of manufacturing the same. 
     Generally, the dual pore sensors described herein are formed by anisotropically etching openings in a monocrystalline silicon substrate or a monocrystalline silicon substrate surface to form at least two fluid reservoirs which are separated from one another by a divider wall disposed therebetween. The width of the barrier wall limits how close the two nanopores of the dual pore sensors may be spaced from one another and is thus determinative of the minimum length of a biopolymer stand that can be co-captured therebetween. 
     Typically, anisotropically etching the two fluid reservoirs forms a divider wall having a triangular or a trapezoidal shape in cross section, see e.g., the trapezoidal shaped cross section of divider wall  314  shown in  FIG. 3D , where the base of the divider wall is wider than the field (upper) surface thereof. In other words, opposing surfaces of the divider wall are sloped to form an angle of less than 90° with a plane of the field surface of the substrate. The sloped surfaces on opposing sides of the divider wall desirably increase stability of the divider wall during manufacturing of the sensor. The added stability allows for the width of the field surface of the divider wall to be narrower, and the fluid reservoirs to be deeper, when compared to a sensor formed from a glass substrate. This is because a divider wall formed in a glass substrate using conventional methods will have vertical sides (i.e., the same wall thickness) along at least a portion the height thereof. Thus, a narrow divider wall formed using conventional methods will undesirably buckle and break as the aspect ratio (height to width ratio) thereof is increased which constrains the manufacturing ability to form narrower walls and deeper reservoirs. 
     Beneficially, the narrower field surface of the divider walls, made possible by the methods set forth herein, allow for closer spacing of the two nanopores and thus allow for sequencing of shorter biopolymer strands. Further, the deeper reservoirs made possible by the methods set forth herein provide a greater cross sectional area for, and thus provide desirably less resistance to, ionic current flow therethrough. 
     Examples of suitable substrates which may be used to form the dual pore sensors herein include those commonly used in semiconductor device manufacturing, such as an N-type or P-type doped monocrystalline silicon wafers, or substrates formed undoped monocrystalline silicon, i.e., intrinsic monocrystalline silicon wafers. In some embodiments, the substrate is a doped or undoped silicon wafer having an epitaxial layer of undoped monocrystalline silicon formed thereon. In some embodiments, the substrate features a layered stack of silicon, an electrically insulating material, such as sapphire or a silicon oxide, and silicon, commonly known as a silicon-on-insulator (SOI) substrate or an SOI wafer. When used, undoped silicon substrates, undoped silicon epitaxial layers, and SOI substrates beneficially reduce undesirable parasitic capacitance in a dual pore sensor formed therefrom when compared to a sensor formed of a doped silicon substrate. 
       FIG. 1A  is a close up sectional view schematically illustrating a portion of a dual pore sensor, formed according to embodiments described herein, which may be used to sequence a biopolymer strand. Here, the dual pore sensor  100  features two fluid reservoirs  102   a, b  and a common chamber  104  each of which, in use, have a conductive fluid, such as an electrolytic fluid, disposed therein. The two fluid reservoirs  102   a, b  are fluidly isolated from one another by a divider wall  105  disposed therebetween. Here, the divider wall  105  of formed of a continuous portion of an underlying monocrystalline silicon substrate  106  or monocrystalline substrate surface which further includes an oxidized surface layer  108  and a silicon nitride layer  110  disposed on the oxidized surface layer  108 . Typically, patterning the underlying monocrystalline silicon substrate  106  forms a triangular or trapezoidal shape in cross section, such as the trapezoidal shaped cross section of the divider wall  314  shown in  FIG. 3 . Herein, oxidizing the surface to form the oxidized surface layer  108  consumes at least a portion of the silicon from the monocrystalline silicon substrate. Thus, in embodiments where the divider wall is formed to have a trapezoidal shape in cross section, oxidizing the monocrystalline silicon surface may result in the triangular cross sectional shape of the continuous portion of an underlying monocrystalline silicon substrate  106  shown in  FIG. 1A . In some embodiments, the oxidized surface layer  108  does not penetrate far enough into the monocrystalline silicon surface to form a triangular shape in cross section. In some embodiments, the monocrystalline silicon surface is not thermally oxidized, although some native oxide may form thereon. 
     The common chamber  104  is separated from the two reservoirs  102   a, b  by a membrane layer  112  having two nanoscale openings, here a first nanopore  114   a  and a second nanopore  114   b , formed therethrough. The first nanopore  114   a  is disposed through a portion of the membrane layer  112  which separates the first reservoir  102   a  from the common chamber  104 . The second nanopore  114   b  is disposed through a portion of the membrane layer  112  which separates the second reservoir  102   b  from the common chamber  104 , and the divider wall separates the first and second reservoirs  102   a, b  from each other. 
     Source electrodes  116   a, b , disposed in each of the fluid reservoirs  102   a, b , respectively, and a common ground electrode  118  disposed in the common chamber  104 , are used to apply independent voltage potentials to each of the fluid reservoirs  102   a, b  V 1 , V 2  as compared to the ground potential of the common chamber to facilitate co-capture of a single biopolymer strand  120 . Once co-capture of the biopolymer strand  120  is achieved by the first and second nanopores  114   a, b , application of competing voltages across the first and second nanopores  114   a, b , i.e., between their electrodes  116   a, b  and the common ground electrode  118  respectively, are used to create a tug-of-war on the biopolymer strand as it travels from the first reservoir  102   a  to the second reservoir  102   b . Ionic current flows are independently measured through each of the nanopores  114   a, b  and the resulting current signal patterns can be used to determine a sequence of the monomer units of the biopolymer strand. 
       FIG. 1B  schematically illustrates trapezoidal cross-section shaped openings  121  formed in a monocrystalline silicon substrate  122  using an anisotropic etch process and a patterned mask layer  128  disposed on the surface thereof. The anisotropic etch process uses inherently differing etch rates for the silicon material of the substrate as between {100} plane surfaces  124  and {111} plane surfaces  126  thereof when exposed to an anisotropic etchant. The actual differing etch rates of the silicon substrate  122  into {100} plane surfaces  124  and {111} plane surfaces  126  depend on the concentration of the etchant in the aqueous solution, the temperature of the aqueous solution, and a concentration of the dopant in the substrate (if any). 
     In some embodiments, the etching process is controlled to where the etch rates of the {111} plane surfaces  126  and the {100} plane surfaces have a ratio between about 1:10 and about 1:200 such as between about 1:10 and about 1:100, for example between about 1:10 and 1:50, or between about 1:25 and 1:75). Examples of suitable anisotropic wet etchants herein include aqueous solutions of potassium hydroxide (KOH), ethylene diamine and pyrocatechol (EPD), ammonium hydroxide (HN 4 OH), hydrazine (N 2 H 4 ), or tetra methyl ammonium hydroxide (TMAH). 
     Typically, a {100} plane at the surface of monocrystalline silicon substrate will meet the {111} plane in the bulk of the substrate to form an angle α of 54.74°. Thus, in embodiments set forth herein sidewalls defining anisotropically etched openings in a monocrystalline silicon substrate will form an angle with a plane of the field surface of the substrate of about 54.74°. 
       FIG. 2  is a flow diagram setting forth a method of forming a dual pore sensor, according to one embodiment.  FIGS. 3A-3L  schematically illustrate various activities of the method  200 , according to one or more embodiments. 
     At activity  201  the method  200  includes providing a pattern in a surface of a substrate. Here, the pattern features two fluid reservoirs recessed from a field of the surface, where the two fluid reservoirs are separated by a barrier wall formed of non-recessed or partly recessed portion of the substrate. In one embodiment, providing the pattern in the surface of the substrate surface includes forming a patterned mask layer on the surface of a substrate and transferring the pattern of the etch mask to the underlying substrate surface using an anisotropic etch process.  FIGS. 3A and 3B  illustrate a substrate  302  having a patterned mask layer  304  disposed thereon.  FIG. 3A  is a schematic plan view of the substrate and mask thereover.  FIG. 3B  is a sectional view of a portion of  FIG. 3A  taken along line A-A. 
     Here, the patterned mask layer  304  is formed of a material which is selective to anisotropic etch compared to the underlying monocrystalline silicon substrate. Examples of suitable mask materials include silicon oxide (Si x O y ) or silicon nitride (Si x N y ). Herein, the mask layer  304  has a thickness of about 100 nm or less, such as about 50 nm or less, or about 30 nm or less. The mask layer  304  material here is patterned using any suitable combination of lithography and material etching patterning methods. The pattern features a first opening  306   a  and a second opening  306   b  disposed through the mask layer  304  which are spaced apart from one another to define a mask wall  308  disposed therebetween. Here, openings  306   a, b  define two sides of a recessed pattern generally surrounded by the masking material and divided by the mask wall  308 , and individual generally circularly cylindrical islands  310  of mask material interspersed in the respective recess. 
     In  FIG. 3A  the two openings  306   a, b  form a generally symmetrical “H” shaped pattern which is bifurcated by the mask wall  308 . In other embodiments, the pattern may be any suitable symmetrical or asymmetrical shape for example an “X” shaped pattern, a “+” shaped pattern, a “K” shaped pattern, or any other desired pattern where the to-be-formed reservoirs will come into close proximity to form the a divider wall having a desired width. 
     In  FIG. 3B  the islands  310   a  are bisected by line A-A are shown in cross section, the islands  310   b  are behind the section defined by line A-A. A width X 1  of the mask wall  308  at the field (upper) surface of the substrate  302  and the amount of material removed from the  111  plane during a subsequent anisotropic etch process determines the minimum spacing between the two nanopores of the dual nanopore sensor. Here, the width X 1  is less than about 300 nm, such less than about 250 nm, less than about 200 nm, or for example less than about 180 nm. The mask layer  304  further includes a plurality of discontinuous features as individual mesas or islands  310  of mask material, distributed within the boundaries defined by the walls of each of the openings  306   a, b.    
     Transferring the mask pattern to the surface of the substrate  302  typically comprises anisotropically etching the monocrystalline silicon thereof by exposing the field surface thereof to an etchant through the openings  306   a, b  of the mask layer  304 . In one embodiment, anisotropically etching the substrate  302  comprises exposing the substrate surface to an anisotropic wet etchant to form first and second reservoirs  312   a, b  (shown in  FIGS. 3C-3D ) each having a base surface which is recessed from the field surface of the substrate to a desired depth D. Here, each of the first and second reservoirs  312   a, b  will form a respective fluidly connected volume in the resulting dual nanopore sensor. After the substrate surface is patterned, the mask layer  304  may be removed therefrom using any suitable method, such as by exposure to an aqueous solution of phosphoric acid. 
       FIG. 3C  is a schematic plan view of the patterned surface of the substrate  302  mask layer  304  is removed.  FIG. 3D  is a schematic sectional view of  FIG. 3C  taken along line B-B. Here, the patterned surface of the substrate  302  features the two fluid reservoirs  312   a, b , which are spaced apart from one another by a divider wall  314 . The fluid reservoirs  312   a, b  each have a maximum depth D 1  of measured in a direction orthogonal the field surface of the substrate  302 . Typically, the maximum depth D 1  is more than 0.1 μm, such as more than 0.5 μm, or more than about 1 μm, for example between about 0.5 μm and about 2 μm. Here, the patterned surface further includes a plurality of support structures  316  corresponding to the locations of the plurality of islands  310  described above. Each of the plurality of support structures  316  have a truncated cone or pyramidal shape which forms a trapezoidal shape in cross-section where the field surfaces of the support structures  316  are narrower than the bases thereof. Here, the widths W 2  of individual support structures  316  at the field surfaces thereof are in a range of between about 0.1 μm and about 5 μm, such as between about 0.5 μm and about 2.5 μm. Individual ones of the plurality of the support structures  316  spaced apart from the walls of first and second openings  306   a, b  and from one another by a distance suitable for supporting portions of a to-be-formed membrane layer which will span the reservoirs  312   a, b . In some embodiments, the support structures have a center to center spacing of 10 μm or less, such as about 7.5 μm or less, or for example about 5 μm or less. 
     Here, the divider wall  314  has a trapezoidal shape in cross section such that opposing surfaces thereof are sloped to form an angle α of 54.74° with a plane of the field surface of the patterned substrate  302 . The width W 1  of the divider wall  314  at the field surface of the substrate  302  is about 200 nm or less, such as 180 nm or less, about 160 nm or less, about 140 nm or less, about 120 nm or less, or about 100 nm or less. In some embodiments, the width W 1  is in the range between about 60 nm and about 140 nm, such as between about 80 nm and about 120 nm. In other embodiments the openings forming the fluid reservoirs  312   a, b , are etched until the divider wall  314  has a triangular shape in cross section. 
     Here, the method  200  further includes forming a dielectric layer on the patterned surface of the substrate  302  by one or both of thermally oxidizing the monocrystalline silicon surface or by depositing a dielectric material thereon. For example, in some embodiments, the method  200  further includes thermally oxidizing the surface of the substrate to form an oxide layer, herein the first dielectric layer  318  (shown in  FIG. 3E ). In some embodiments, the silicon surface is oxidized to provide a first dielectric layer  318  having a thickness of more than about 5 nm, such as more than about 10 nm, more than about 20 nm, or more than about 30 nm. In some embodiments, the silicon surface is oxidized to provide a first dielectric layer  318  having a thickness of between about 20 nm and about 80 nm. Typically, thermal oxidation comprises exposing the substrate  302  to steam or molecular oxygen (O 2 ) in a furnace at a temperature between about 800° C. and about 1200° C. Because thermal oxide incorporates silicon consumed from the substrate  302  with supplied oxygen, about 44% of the thickness of the first dielectric layer  318  will lie below the original silicon surface and about 56% of the thickness of the first dielectric layer  318  will extend thereabove. Thus, thermally oxidizing the silicon surface to form the first dielectric layer  318  will increase the width of the wall by more than about 1.12 times the thickness of the resulting thermal oxide. In some embodiments, the silicon surface is thermally oxidized to a depth where the portion forming the divider wall has a triangular cross sectional shape. In some embodiments, the silicon surface is thermally oxidized to a depth where the portion forming the divider wall maintains its trapezoidal cross sectional shape. 
     In some embodiments, the method  200  includes depositing a dielectric material, such as the second dielectric layer  320  ( FIG. 3E ) on the patterned surface to cover and thus line the surfaces of the two fluid reservoirs  312   a, b  and the field. Here, the second dielectric layer  320  comprises any suitable dielectric material, such as a silicon oxide (Si x O y ), a silicon nitride (Si x N y ), a silicon oxynitride (SiO x N y ), or an oxide, nitride, or oxynitride of Group III, Group IV, Lanthanide series elements, combinations thereof, or layered stacks of two or more thereof. For example, in some embodiments, the second dielectric layer  320  comprises aluminum oxide (Al 2 O 3 ), aluminum nitride (AlN), titanium oxide (TiO), titanium nitride (TiN), tantalum oxide (Ta 2 O 5 ), tantalum nitride (TaN), or combinations thereof. In some embodiments, the second dielectric layer  320  comprises amorphous silicon. 
     Beneficially, the second dielectric layer  320  prevents or substantially reduces charges from accumulating in the monocrystalline silicon substrate  302  during high frequency nucleotide detection. Thus, the second dielectric layer  320  substantially reduces undesirable background noise to improve the detection resolution of the dual pore sensor. Here, the second dielectric layer  320  is deposited to a thickness of less than about 100 nm, such as less than about 80 nm, less than about 60 nm, or for example between about 20 nm and about 100 nm. Depositing the second dielectric layer  320  increases the width of the wall by more than about 2 times the thickness of the second dielectric layer  320 . 
     Typically, the sloped surfaces of the first or second dielectric layer  318 ,  320  disposed on opposing sides of the divider wall  314  will form an angle θ with the plane of the field surface of the substrate  302  having one or both of the dielectric layer  318 ,  320  disposed thereon. Here, the angle θ may be the same as the angle α of about 54.74° or may vary to account for non-uniform oxidation of the substrate  302  to form the first dielectric layer  318  and, or, non-conformal deposition of the second dielectric layer  320 . For example, in some embodiments the sloped surfaces of the first or second dielectric layer  318 ,  320  form an angle θ in a range of about 54.74°+/−5°, or about 54.74°+/−2.5°, or about 54.74°+/−1°. 
     The second dielectric layer  320  may serve as a CMP stop layer in subsequent planarization operations and, or electrically insulate conductive fluid in the fluid reservoirs  312   a, b  from the monocrystalline silicon substrate  302  disposed therebelow. In some embodiments, the method  200  includes one but not both of oxidizing the patterned surface of the substrate  302  to form the first dielectric layer  318  or depositing the second dielectric layer  320 . For example, in some embodiments, the patterned surface of the monocrystalline silicon substrate  302  is not thermally oxidized before the second dielectric layer  320  is deposited thereon, although at least some native oxide growth is to be expected. In embodiments that do not include depositing the second dielectric layer  320 , the first dielectric layer  318  may serve as a CMP stop layer in a subsequent planarization operation. 
     At activity  202  the method  200  includes filling the two fluid reservoirs  312   a, b  with a sacrificial material  322 . In some embodiments, filling the two fluid reservoirs  312   a, b , with a sacrificial material  322  includes depositing a layer of sacrificial material  322  onto the patterned substrate  302 , e.g., onto the first dielectric layer  318  or the second dielectric layer  320  ( FIG. 3F ). In those embodiments, the method further includes removing the sacrificial material  322  from over a field surface of the second dielectric layer  320  ( FIG. 3G ) to leave the portions of the second dielectric layer  320  over each of the dividing walls exposed. Typically, removing the sacrificial material  322  from the field surface of the second dielectric layer  320  comprises planarizing a surface of the substrate using a chemical mechanical planarization (CMP) process. The planarized surface of the substrate, including the planarized surfaces of the sacrificial material  322  disposed in the fluid reservoirs  312   a, b  (shown in  FIG. 3E ), will provide structural support for a subsequently deposited membrane layer. A suitable sacrificial material will have a high etch rate and CMP removal rate selectivity to the underlying second dielectric layer  320  and a high etch rate selectivity to the material of the membrane layer  112  to-be-formed thereover. Examples of suitable sacrificial materials include phosphosilicate glass (PSG), borophosphosilicate glass (BPSG), polysilicon, amorphous Si, aluminum, carbon-based films, and polymers such as polyimide. 
     At activity  203  the method  200  includes depositing a membrane layer  324 . Here, the membrane layer  324  is deposited onto the field surface of the second dielectric layer  320  and onto the planarized sacrificial material  322  disposed in the fluid reservoirs  312   a, b . In some embodiments, the membrane layer  324  is formed of silicon nitride. In other embodiments, the membrane layer is formed of another suitable dielectric material, such as any of the materials set forth above as suitable for the second dielectric layer  320 . Typically, the membrane layer  324  is deposited to a thickness of less than about 200 nm, such as less than about 100 nm, less than about 60 nm, for example less than about 50 nm, or between about 10 nm and about 50 nm, such as between about 20 nm and about 40 nm. 
     At activity  205  the method  200  includes removing the sacrificial material  322  from the two fluid reservoirs  312   a, b . In one embodiment, removing the sacrificial material  322  includes patterning the membrane layer  324  to form a plurality of vent openings  326  therethrough and removing the sacrificial material  322  through the plurality of vent openings  326 . The membrane layer  324  may be patterned using any suitable combination of lithography and material etching patterning methods such as forming a patternable mask layer over the membrane layer  324 , patterning the mask layer to form openings corresponding in size and location to the locations of the vent openings  326  using photolithographic techniques, and then etching the portions of the membrane layer  324  exposed by the openings through the mask layer to form the vent openings  326  through the membrane layer  324 . 
     Here, individual ones of the plurality of vent openings  326  have a diameter of less than about 500 nm, less than about 100 nm, or for example less than about 50 nm. In some embodiments, individual ones of the plurality of vent openings  326  have a diameter of between about 1 nm and about 500 nm, such as between about 1 nm and about 100 nm, between about 1 nm and about 50 nm, or for example between about 10 nm and about 40 nm. In some embodiments, individual ones of the plurality of vent openings  326  have a center to center spacing from a vent opening  326  disposed adjacent thereto of less than about 500 nm, such as less than about 300 nm, or less than about 100 nm. The plurality of vent openings  326  may from any desirable pattern suitable for venting volatilized or dissolved sacrificial material  322  disposed in the fluid reservoirs  312   a, b  therethrough in a subsequent sacrificial material removal step including the irregularly spaced pattern shown in  FIG. 3H . 
     In one embodiment, the sacrificial material  322  is removed through the vent openings  326  using a plasma based dry etch process. For example, in one embodiment the sacrificial material  322  is exposed through the plurality of vent openings  326  to the plasma activated radical species of a suitable etchant, such as the radial species of a halogen based gas, e.g., a fluorine or chlorine based gas. An exemplary system which may be used to remove the sacrificial material  322  from the fluid reservoirs  312   a, b  is the Producer® Selectra™ Etch system commercially available from Applied Materials, Inc., of Santa Clara, Calif. as well as suitable systems from other manufacturers. 
     In another embodiment, removing the sacrificial material  322  includes exposing the sacrificial material  322 , through the vent openings  326 , to an etchant having a relativity high etch selectivity to the material or materials used to form the second dielectric layer  320  and the membrane layer  324 . Examples of suitable etchants include TMAH, NH4OH, aqueous HF solutions, and buffered aqueous HF solutions such as an aqueous solution of HF and NH4F, and anhydrous HF. Etch byproducts are then removed from the fluid reservoirs  322   a, b  by rinsing and drying the substrate. In some embodiments the etch byproducts are removed by rinsing the substrate with deionized water before drying the substrate using N 2  gas or an isopropyl alcohol (IPA) and N 2  gas mixture. In other embodiments, such as in embodiments using anhydrous HF, removing remaining etch byproducts includes heating the substrate to a temperature of more than about 100° C. in a vacuum environment of less than about 40 Torr. 
     At activity  205  the method  200  includes patterning two nanopores  328   a, b  through the membrane layer  324 . The nanopores  328   a, b  may be patterned using any suitable method. In one embodiment, the nanopores  328   a, b  are patterned using the same or a similar process to the process used to form the vent openings  326  as described above. For example, in some embodiments, the vent openings  326  and the nanopores  328   a, b  are formed in the same lithography and material etching sequence. In other embodiments, the vent openings  326  and the nanopores  328   a, b  are formed in sequential lithography and material etching sequences of any order. In other embodiments, the nanopores  328   a, b  are formed in a lithography and material etching sequence which is separated from the lithography and material etching sequence used to form the vent openings  326  by another processing operation. For example, in some embodiments the nanopores  328   a, b  are formed after the sacrificial material  322  is removed through the vent openings  326  or after a common chamber is patterned as described in activity  206  below. 
     Here, the two nanopores  328   a, b  are formed through respective portions of the membrane layer  324  disposed over each of the fluid reservoirs  312  a, b and thus are positioned on either side of the divider wall  314  proximate thereto. Typically, each of the nanopores  328   a, b  have a diameter of less than about 100 nm, such as less than about 50 nm between about 0.1 nm and about 100 nm, or between about 0.1 nm and about 50 nm. Here, the nanopores  328   a, b  are spaced apart from one another by a distance X 2  of less than about 600 nm, such as less than about 550 nm, less than about 500 nm, less than about 450 nm, less than about 400 nm, or in some embodiments, less than about 300 nm. 
     At activity  206  the method  200  includes patterning one or more fluid ports  338  and a common chamber  334  ( FIG. 3J ). In one embodiment, patterning the one or more fluid ports  338  and the common chamber  334  forming openings in an overcoat layer  330  disposed on the patterned membrane layer  324 . Here, the overcoat layer  330  seals the vent openings  326  in the membrane layer  324  where fluid access to the reservoirs  332   a, b  disposed therebeneath is not desired. The one or more fluid ports  338  provide fluid access to the fluid reservoirs  332   a, b  to facilitate the introduction of electrolytic fluid and biopolymer samples thereinto. The overcoat layer  330  may be formed using any suitable material and method which minimizes penetration of the overcoat material into the vent openings  326 . Thus, the material and method chosen to deposit the overcoat layer  330  should prevent undesirable filling of the fluid reservoirs  332   a, b  therewith through the vent openings  326 . 
     In one embodiment, the overcoat layer  330  is formed by spin coating a polymer precursor onto the patterned membrane layer  324  and curing the polymer precursor by exposure to thermal or electromagnetic radiation. In some embodiments, the fluid ports  338  and the common chamber  334  areas are then etched through the cured polymer using a lithography-etch processing sequence. In other embodiments, the polymer precursor is photosensitive, such as a photosensitive polyimide precursor or benzocyclobutene (BCB), and the desired pattern is exposed directly thereon. Unexposed photosensitive polymer precursor is then removed from the substrate to form the fluid ports  338  and the common chamber  334  areas. Herein, the fluid port  338  and the common chamber  334  areas may be formed at the same time, sequentially, or in processing operations separated by intervening processing activities. 
     In another embodiment, the overcoat layer  330  comprises a polymer film layer, such as a polyimide film, which is laminated onto the surface of the membrane layer  324  before or after the fluid port  338  and the common chamber  334  areas are formed (patterned) therethrough. 
       FIG. 3J  is a schematic plan view of a dual pore sensor  300 , formed according to embodiments described herein, which may be used in place of the dual pore sensor  100  described in  FIG. 1A .  FIG. 3K  is a sectional view of a portion of  FIG. 3J  taken along line D-D. Here, the dual pore sensor  300  features a patterned substrate  301  and the membrane layer  324  disposed on the patterned substrate  301 . The pattern includes two recessed regions separated by the divider wall  314 . Each of the two recessed regions have one or more base surfaces  303  which are substantially parallel to a plane of the field (upper) surface of the patterned substrate  301 . The base surfaces  303  and one or more sidewalls  305  of the each of the recessed regions (shown in phantom in  FIG. 3J ), the membrane layer  324 , and the divider wall  314  (having one or both dielectric layers  318 ,  320  disposed thereon) collectively define the first fluid reservoir  332   a  and the second fluid reservoir  332   b  respectively. 
     Here, the membrane layer  324  is spaced apart from the one or more base surfaces  303  of the recessed regions by a distance D 2  of more than about 0.5 μm, such as more than about 1 μm, more than about 1.5 μm, or more than about 2 μm. The surfaces of the recessed regions and the divider wall  314  are lined with one or both of the first or second dielectric layer  318 ,  320 . A first nanopore  328   a  is disposed through a portion of the membrane layer  324  disposed over the first fluid reservoir  332   a  and a second nanopore  328   b  is disposed through a portion of the membrane layer  324  disposed over the second fluid reservoir  332   b . In some embodiments, membrane layer  334  has a plurality of vent openings  326  formed therethrough which are sealed with a overcoat layer  330  disposed thereover. The overcoat layer  330  includes openings disposed therethrough to define the common chamber  334  and the one or more fluid ports  338  disposed over each of the respective fluid reservoirs  332   a, b . The common chamber  334  is in fluid communication with each of the fluid reservoirs  332   a, b , through respective nanopores  328   a, b.    
     Here, the reservoir facing surface of the membrane layer  324  is substantially planer and is parallel to the field surface of the patterned substrate  301 . In some embodiments, the membrane layer  324  is spaced apart from the base surfaces  303  of the recessed regions by the plurality of support structures  316  (and the dielectric liner disposed thereon). Typically, individual ones of the plurality of support structures  316  have a trapezoidal shape in cross section. For example, herein surfaces of the one or both of the plurality of support structures  316  and the divider wall  314  are sloped to form an angle θ with a reservoir  332   a, b , facing surface of the membrane layer  324  of less than 90°, such as less than about 60°, or with the range of about 54.74°+/−5°, or about 54.74°+/−2.5°, or 54.74°+/−1°, for example about 54.74°. 
     In some embodiments, a ratio of the depth D 2  of the recessed regions to the nanopore spacing X 2  (described in  FIG. 3I ) is more than about 1:1, such as more than about 2:1, more than about 3:1, more than about 4:1, or for example more than about 5:1. Here, the depth D 2  is measured from a plane of the field surface of the patterned substrate  301  to the base surfaces  303  of the fluid reservoirs  312   a, b , i.e., the distance between reservoir facing surfaces of the membrane layer  324  and the base surfaces  303 . In some embodiments, the dual pore sensor  300  further includes electrodes disposed in each of the fluid reservoirs  332   a, b  and the common chamber  334 , such as the electrodes  116   a, b  and  118  described in  FIG. 1A . 
     In some embodiments, the method  200  further includes forming a vent opening extension layer  332  (shown in  FIG. 3L ) on the membrane layer  324  before removing the sacrificial material  322  from the fluid reservoirs at activity  208 . Forming the vent opening extension layer  332  before removing the sacrificial material  322  may prevent damage to, e.g., collapse of, the fragile underlying membrane layer  324  when the overcoat layer  330  is formed thereon. In those embodiments, the vent opening extension layer  332  may be formed of the same material and methods which are suitable for forming the subsequent overcoat layer  330  and are set forth in the description of activity  208 . Once the vent opening extension layer  332  is deposited onto the membrane layer a plurality of openings  340  are formed therethrough. Each of the plurality of openings  340  are coaxially disposed and/or in fluid registration with a corresponding vent opening extension layer  332  opening  326  in the membrane layer  324 . Examples of suitable methods of forming the plurality of openings  340  include lithography-etch processing sequences and direct exposure of a photosensitive polymer precursor in embodiments where the opening extension layer  332  is formed therefrom. In embodiments which include the vent opening extension layer  332  one or both of the fluid ports and common chamber opening are further formed through the vent opening extension layer to expose the membrane layer disposed there beneath. 
     In some embodiments, the dual pore sensor  300  described in  FIGS. 3J-3K  further includes the vent opening extension layer  332  described above in  FIG. 3L . 
     In another embodiment, the substrate is a silicon on insulator (SOI) substrate  402  (shown in  FIG. 4A ) featuring first and second (monocrystalline) silicon layers  402   a  and  402   c  and an electrical insulator layer  402   b , such as a sapphire layer or a silicon oxide layer (Si x O y ), interposed therebetween. In this embodiment, the surface of the substrate  402 , i.e. the second silicon layer  402   c , is patterned using one or a combination of embodiments of the method  200  set forth above to form a patterned substrate  405  ( FIG. 4B ). The pattern comprises two fluid reservoirs  412   a, b , a divider wall  414  having a width W 4  at the field surface thereof, and a plurality of structural supports  416  formed in the second silicon layer  402   c . The patterned second silicon layer  402   c  is thermally oxidized to the depth of the electrical insulator layer  402   b  disposed there beneath and a dual pore sensor may be formed therefrom using activities  202 - 208  of the method  200  or alternative embodiments thereof. 
     In some embodiments, the method  200  above includes forming the pattern in the second silicon layer  402   c  and thermally oxidizing the second silicon layer  402   c  to the depth of the electrical insulator layer  402   b . In some embodiments, the patterned second silicon layer  402   c  is not oxidized to the depth of the electrical insulator layer  402   b . For example, in some embodiments the patterned second silicon layer  402   c  is thermally oxidized to a depth of less than about 100 μm, such as less than about 50 μm, less than 25 μm, or for example less than about 10 μm. 
     In some embodiments, the dual pore sensor  300  described in  FIGS. 3J-3K  features one or both of the patterned substrate  405  in place of the patterned substrate  301  and the vent opening extension layer  332 . In some embodiments, the patterned substrate  405  further includes a dielectric liner, such as the second dielectric  320  described above, deposited thereon. 
     Typically, the methods provided herein are used to simultaneously manufacture a plurality of dual pore sensors on a single substrate, such as the single wafer substrate  500  shown in  FIG. 5 . The wafer substrate  500  is then singulated into individual dies to provide a plurality of dual pore sensors  300 . 
     Exemplary dimensions of a sensor  300  formed using the methods set forth herein is less than about 20 mm per side, such as less than about 15 mm, or less than about 10 mm, or for example between about 1 mm and about 20 mm. In some embodiments a width of a singulated sensor formed using the embodiments set forth herein is between about 1 mm and about 100 mm. 
     The dual pore sensors provided herein may include any one or combination of the features described above in  FIGS. 1A, 3J-3K, 3L, and 4B , including alternate embodiments thereof. The dual pore sensors provided herein may be singulated or may comprise one of a plurality of dual pore sensors formed on a single wafer substrate, such as the single wafer substrate  500  described in  FIG. 5 . 
     Beneficially, the methods described herein allow for high volume manufacturing, and improved quality, repeatability, and manufacturing costs of a dual pore sensor. Further, the manufacturing methods described allow for interpore spacing of 300 nm or less to beneficially increase the number of relativity shorter biopolymer strands which may be sequenced using a dual pore sensor. 
     While the foregoing is directed to embodiments of the present disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.