Abstract:
A system and method employ at least one semiconductor device, or an arrangement of insulating and metal layers, having at least one detecting region which can include, for example, a recess or opening therein, for detecting a charge representative of a component of a polymer, such as a nucleic acid strand proximate to the detecting region. A method for manufacturing forms such a semiconductor device. The system and method can be used for sequencing individual nucleotides or bases of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The detecting region permits a current to pass between the two doped regions in response to the presence of the component of the polymer, such as a base of a DNA or RNA strand. The current has characteristics representative of the component of the polymer, such as characteristics representative of the detected base of the DNA or RNA strand.

Description:
CROSS-REFERENCE TO RELATED APPLICATION(S) 
     This application is a continuation of U.S. patent application Ser. No. 11/301,259, filed on Dec. 13, 2005, which is a division of U.S. patent application Ser. No. 10/258,439, filed on Oct. 24, 2002, now U.S. Pat. No. 7,001,792, which is the U.S. national stage of International patent Application No. PCT/US01/13101, filed on Apr. 24, 2001. International Patent Application No. PCT/US01/13101 claims the benefit of U.S. Provisional Patent Application Ser. No. 60/259,584, filed on Jan. 4, 2001, of U.S. patent application Ser. No. 09/653,543, filed on Aug. 31, 2000, now U.S. Pat. No. 6,413,792, of U.S. Provisional Patent Application Ser. No. 60/199,130, filed on Apr. 24, 2000, and of U.S. Provisional Patent Application Ser. No. 60/217,681, filed on Jul. 12, 2000. U.S. patent application Ser. No. 09/653,543 claims the benefit of U.S. Provisional Patent Application Ser. No. 60/199,130, filed on Apr. 24, 2000, and of U.S. Provisional Patent Application Ser. No. 60/217,681, filed on Jul. 12, 2000. The entire contents of all of said prior applications are incorporated herein by reference. 
    
    
     BACKGROUND 
     1. Field of the Disclosure 
     The present invention relates to a system and method employing a semiconductor device having a detecting region for identifying the individual mers of long-chain polymers, such as carbohydrates and proteins, as well as individual bases of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and a method for making the semiconductor device. More particularly, the present invention relates to a system and method employing a semiconductor device, similar to a field-effect transistor device, capable of identifying the bases of a DNA/RNA strand to thus enable sequencing of the strand to be performed. 
     2. Description of the Related Art 
     DNA consists of two very long, helical polynucleotide chains coiled around a common axis. The two strands of the double helix run in opposite directions. The two strands are held together by hydrogen bonds between pairs of bases, consisting of adenine (A), thymine (T), guanine (G), and cytosine (C). Adenine is always paired with thymine, and guanine is always paired with cytosine. Hence, one strand of a double helix is the complement of the other. 
     Genetic information is encoded in the precise sequence of bases along a DNA strand. In normal cells, genetic information is passed from DNA to RNA. Most RNA molecules are single stranded but many contain extensive double helical regions that arise from the folding of the chain into hairpin-like structures. 
     Mapping the DNA sequence is part of a new era of genetic-based medicine embodied by the Human Genome Project. Through the efforts of this project, one day doctors will be able to tailor treatment to individuals based upon their genetic composition, and possibly even correct genetic flaws before birth. However, to accomplish this task it will be necessary to sequence each individual&#39;s DNA. Although the human genome sequence variation is approximately 0.1%, this small variation is critical to understanding a person&#39;s predisposition to various ailments. In the near future, it is conceivable that medicine will be “DNA personalized,” and a physician will order sequence information just as readily as a cholesterol test is ordered today. Thus, to allow such advances to be in used in everyday life, a faster and more economical method of DNA sequencing is needed. 
     One method of performing DNA sequencing is disclosed in U.S. Pat. No. 5,653,939, the entire content of which is incorporated herein by reference. This method employs a monolithic array of test sites formed on a substrate, such as a semiconductor substrate. Each test site includes probes which are adapted to bond with a predetermined target molecular structure. The bonding of a molecular structure to the probe at a test site changes the electrical, mechanical and optical properties of the test site. Therefore, when a signal is applied to the test sites, the electrical, mechanical, or optical properties of each test site can be measured to determine which probes have bonded with their respective target molecular structure. However, this method is disadvantageous because the array of test sites is complicated to manufacture, and requires the use of multiple probes for detecting different types of target molecular structures. 
     Another method of sequencing is known as gel electrophoresis. In this technology, the DNA is stripped down to a single strand and exposed to a chemical that destroys one of the four nucleotides, for example A, thus producing a strand that has a random distribution of DNA fragments ending in A and labeled at the opposite end. The same procedure is repeated for the other three remaining bases. The DNA fragments are separated by gel electrophoresis according to length. The lengths show the distances from the labeled end to the known bases, and if there are no gaps in coverage, the original DNA strand fragment sequence is determined. 
     This method of DNA sequencing has many drawbacks associated with it. This technique only allows readings of approximately 500 bases, since a DNA strand containing more bases would “ball” up and not be able to be read properly. Also, as strand length increases, the resolution in the length determination decreases rapidly, which also limits analysis of strands to a length of 500 bases. In addition, gel electrophoresis is very slow and not a workable solution for the task of sequencing the genomes of complex organisms. Furthermore, the preparation before and analysis following electrophoresis is inherently expensive and time consuming Therefore, a need exists for a faster, consistent and more economical means for DNA sequencing. 
     Another approach for sequencing DNA is described in U.S. Pat. Nos. 5,795,782 and 6,015,714, the entire contents of which are incorporated herein by reference. In this technique, two pools of liquid are separated by a biological membrane with an alpha hemolysin pore. As the DNA traverses the membrane, an ionic current through the pore is blocked. Experiments have shown that the length of time during which the ionic current through the pore is blocked is proportional to the length of the DNA fragment. In addition, the amount of blockage and the velocity depend upon which bases are in the narrowest portion of the pore. Thus, there is the potential to determine the base sequence from these phenomena. 
     Among the problems with this technique are that individual nucleotides cannot, as yet, be distinguished. Also, the spatial orientation of the individual nucleotides is difficult to discern. Further, the electrodes measuring the charge flow are a considerable distance from the pore, which adversely affects the accuracy of the measurements. This is largely because of the inherent capacitance of the current-sensing electrodes and the large statistical variation in sensing the small amounts of current. Furthermore, the inherent shot noise and other noise sources distort the signal, incurring additional error. Therefore, a need exists for a more sensitive detection system which discriminates among the bases as they pass through the sequencer. 
     SUMMARY 
     An object of the present invention is to provide a system and method for accurately and effectively identifying individual bases of DNA or RNA. 
     Another object of the present invention is to provide a system and method employing a semiconductor device for sequencing individual bases of DNA or RNA. 
     A further object of the present invention is to provide a method for manufacturing a semiconductor-based DNA or RNA sequencing device. 
     Another object of the present invention is to provide a system and method for accurately and effectively identifying the individual mers of long-chain polymers, such as carbohydrates or proteins, as well as measuring the lengths of the long-chain polymers. 
     Still another object of the present invention is to provide a system and method employing a semiconductor-based device having an opening therein, for accurately and effectively identifying bases of DNA or RNA by measuring charge at a location where the DNA or RNA molecules traverse the opening in the sequencer, to thus eliminate or at least minimize the effects of shot noise and other noise sources associated with the random movement of the DNA or RNA molecules through the opening. 
     These and other objects of the invention are substantially achieved by providing a system for detecting at least one polymer, comprising at least one semiconductor device, or an arrangement of insulating and metal layers, having at least one detecting region which is adapted to detect a charge representative of a component of the polymer proximate to the detecting region. The component can include a base in a nucleic acid strand, so that the detecting region is adapted to detect the charge which is representative of the base in the nucleic acid strand. The detecting region is further adapted to generate a signal representative of the detected charge. Also, the detecting region can include a region of the semiconductor device defining a recess in the semiconductor device, or an opening in the semiconductor device having a cross-section sufficient to enable the polymer to enter the opening, so that the detecting region detects the charge of the component in the opening. Furthermore, the semiconductor device preferably further includes at least two doped regions, and the detecting region can pass a current between the two doped regions in response to a presence of the component proximate to the detecting region. 
     The above and other objects of the invention are also substantially achieved by providing a method for detecting at least one polymer, comprising the steps of positioning a portion of the polymer proximate to a detecting region of at least one semiconductor device, and detecting at the detecting region a charge representative of a component of the polymer proximate to the detecting region. The component can include a base in a nucleic acid strand, so that the detecting step detects a charge representative of the base. The method further comprises the step of generating at the detecting region a signal representative of the detected charge. The detecting region can include a region of the semiconductor device defining a recess in the semiconductor device, or an opening in the semiconductor device having a cross-section sufficient to enable the polymer to enter the opening, so that the detecting step detects the charge of the component in the recess or opening. Furthermore, the semiconductor device can further include at least two doped regions, so that the method can further include the step of passing a current between the two doped regions in response to a presence of the component proximate to the detecting region. 
     The above and other objects of the invention are further substantially achieved by providing a method for manufacturing a device for detecting a polymer, comprising the steps of providing a semiconductor structure comprising at least one semiconductor layer, and creating a detecting region in the semiconductor structure, such that the detecting region is adapted to detect a charge representative of a component of the polymer proximate to the detecting region. The component can include a base in a nucleic acid strand, and the detecting region can be created to detect a charge representative of the base in the nucleic acid strand. The method can further include the step of creating a recess in the semiconductor structure, or creating an opening in the semiconductor structure having a cross-section sufficient to enable a portion of the polymer to pass therethrough, and being positioned in relation to the detecting region such that the detecting region is adapted to detect the charge representative of the component in the recess or opening. The method can further include the step of forming an insulating layer on a wall of the semiconductor layer having the opening to decrease the cross-section of the opening. Furthermore, the method can include the step of creating at least two doped regions in the semiconductor layer which are positioned with respect to the detecting region such that the detecting region is adapted to pass a current between the doped regions in response to the component of the polymer proximate to the detecting region. The doped regions can be separated by a portion of the semiconductor layer having a different doping, and can be created as a stack of doped regions, each having a first doping and being separated by a layer having a second doping. The doped regions can include either a p-type or an n-type doping. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       These and other objects, advantages and novel features of the invention will be more readily appreciated from the following detailed description when read in conjunction with the accompanying drawings, in which: 
         FIG. 1  illustrates a system for performing DNA or RNA sequencing comprising a DNA or RNA sequencer constructed in accordance with an embodiment of the present invention; 
         FIG. 2  illustrates a top view of the DNA or RNA sequencer shown in  FIG. 1 ; 
         FIG. 3  is a graph showing an example of the waveform, representing the current detected by a current detector in the system shown in  FIG. 1  as the adenine (A), thymine (T), guanine (G), and cytosine (C) bases of a DNA or RNA sequence pass through the DNA or RNA sequencer; 
         FIG. 4  illustrates a cross-sectional view of a silicon-on-insulator (SOI) substrate from which a DNA or RNA sequencer as shown in  FIG. 1  is fabricated in accordance with an embodiment of the present invention; 
         FIG. 5  illustrates a cross-sectional view of the SOI substrate shown in  FIG. 5  having shallow and deep n-type regions formed in the silicon layer, and a portion of the substrate etched away; 
         FIG. 6  illustrates a cross-sectional view of the SOI substrate shown in  FIG. 5  in which a portion of the insulator has been etched away and another shallow n-type region has been formed in the silicon layer; 
         FIG. 7  illustrates a cross-sectional view of the SOI substrate having an opening etched therethrough; 
         FIG. 8  illustrates a top view of the SOI substrate as shown in  FIG. 7 ; 
         FIG. 9  illustrates a cross-sectional view of the SOI substrate shown in  FIG. 7  having an oxidation layer formed on the silicon layer and on the walls forming the opening therein; 
         FIG. 10  illustrates a top view of the SOI substrate as shown in  FIG. 9 ; 
         FIG. 11  illustrates a detailed cross-sectional view of the SOI substrate shown in  FIG. 7  having an oxidation layer formed on the silicon layer and on the walls forming the opening therein; 
         FIG. 12  illustrates a top view of the SOI substrate shown in  FIG. 11 ; 
         FIG. 13  illustrates a detailed cross-sectional view of an exemplary configuration of the opening in SOI substrate shown in  FIG. 7 ; 
         FIG. 14  illustrates a top view of the opening shown in  FIG. 13 ; 
         FIG. 15  illustrates a cross-sectional view of the SOI substrate as shown in  FIG. 9  having holes etched in the oxidation layer and metal contacts formed over the holes to contact the shallow and deep n-type regions, respectively; 
         FIG. 16  illustrates a cross-sectional view of the DNA or RNA sequencer shown in  FIG. 1  having been fabricated in accordance with the manufacturing steps shown in  FIGS. 4-15 ; 
         FIG. 17  illustrates a top view of a DNA or RNA sequencer having multiple detectors formed by multiple n-type regions according to another embodiment of the present invention; 
         FIG. 18  illustrates a cross-sectional view of a DNA or RNA sequencer according to another embodiment of the present invention; 
         FIG. 19  illustrates a cross-sectional view of a DNA or RNA sequencer according to a further embodiment of the present invention; 
         FIG. 20  illustrates a cross-sectional view of a DNA or RNA sequencer according to a further embodiment of the present invention; 
         FIG. 21  illustrates a top view of the DNA or RNA sequencer shown in  FIG. 20 ; 
         FIG. 22  is a conceptual block diagram illustrating an example of a matrix arrangement of DNA or RNA sequencers; 
         FIG. 23  is a cross-sectional view of a sequencer having a metal layer in place of a middle semiconductor layer to achieve electron tunneling; 
         FIG. 24  illustrates a detailed cross-sectional view of another exemplary configuration of the opening in SOI substrate shown in  FIG. 7 ; 
         FIG. 25  illustrates a top view of the opening shown in  FIG. 24 ; 
         FIG. 26  illustrates a cross-sectional view of a multi-opening sequencer used with separate liquid regions; 
         FIGS. 27A and 27B  are images of photographs of opening patterns formed in a semiconductor structure; and 
         FIGS. 28A and 28B  are images of photographs of opening patterns forms in a semiconductor structure. 
     
    
    
     DETAILED DESCRIPTION 
       FIGS. 1 and 2  illustrate a system  100  for detecting the presence of a polymer, such as DNA or RNA, a protein or carbohydrate, or a long chain polymer such as petroleum, and more preferably, for identifying the individual mers of the polymer or long chain polymer, as well as the length of the polymer or long chain polymer. The system  100  is preferably adaptable for performing sequencing of nucleic acids, such as DNA or RNA sequencing, according to an embodiment of the present invention. Accordingly, for purposes of this description, the system  100  will be discussed in relation to nucleic acid sequencing. 
     The system  100  includes a nucleic acid sequencing device  102  which, as described in more detail below, is a semiconductor device. Specifically, the nucleic acid sequencing device  102  resembles a field-effect transistor, such as a MOSFET, in that it includes two doped regions, a drain region  104  and a source region  106 . However, unlike a MOSFET, the nucleic acid sequencing device does not include a gate region for reasons discussed below. 
     The nucleic acid sequencing device  102  is disposed in a container  108  that includes a liquid  110  such as water, gel, a buffer solution such as KCL, or any other suitable solution. It is important to note that the solution  110  can be an insulating medium, such as oil, or any other suitable insulating medium. In addition, the container  108  does not need to include a medium such as a liquid. Rather, the container  108  can be sealed and evacuated to create a vacuum in which nucleic acid sequencing device  102  is disposed. Also, although  FIG. 1  shows only a single nucleic acid sequencing device  102  in the container  108  for exemplary purposes, the container can include multiple nucleic acid sequencing devices  102  for performing multiple DNA sequencing measurements in parallel. 
     The liquid  110  or other medium or vacuum in container  108  includes the nucleic acid strands or portions of nucleic acid strands  111  to be sequenced by nucleic acid sequencing device  102 . As further shown, voltage source  112 , such as a direct current voltage source, is coupled in series with a current meter  114  by leads  116  across drain and source regions  104  and  106 , respectively. In this example, the positive lead of voltage source  112  is coupled to the drain region  104  while the negative lead of voltage source  112  is coupled via the current meter  114  to source region  106 . 
     The voltage potential applied across drain and source regions  104  and  106  of nucleic acid sequencing device  102  can be small, for example, about 100 mV, which is sufficient to create a gradient across drain and source regions  104  and  106 , to draw the nucleic acid strands into opening  118  of the nucleic acid sequencing device  102 . That is, the nucleic acid strands  111  move through the opening  118  because of the local gradient. Alternatively or in addition, the liquid can include an ionic solution. In this event, the local gradient causes the ions in the solution to flow through the opening  118 , which assists the nucleic acid strands  111 , such as DNA or RNA, to move through the opening  118  as well. 
     Additional electrodes  113  and  115  positioned in the medium  110  and connected to additional voltage sources  117  and  121  would further facilitate the movement of the nucleic acid strands towards the opening  118 . In other words, the external electrodes  113  and  115  are used to apply an electric field within the medium  110 . This field causes all of the charged particles, including the nucleic acid strand  111 , to flow either toward the opening  118  or away from the opening  118 . Thus electrodes  113  and  115  are used as a means to steer the nucleic acid strands  111  into or out of the opening  118 . In order to connect voltage sources  112  and  117  to the nucleic acid sequencer  102 , metal contacts  123  are coupled to the n-type doped region  128  and  130 , described in more detail below. The electrodes  113  and  115  could also provide a high frequency voltage which is superimposed on the DC voltage by an alternating voltage source  125 . This high frequency voltage, which can have a frequency in the radio frequency range, such as the megahertz range (e.g., 10 MHz), causes the nucleic acid strand  111  and ions to oscillate. This oscillation makes passage of the nucleic acid strand  111  through the opening  118  smoother, in a manner similar to shaking a salt shaker to enable the salt grains to pass through the openings in the shaker. Alternatively, a device  127 , such as an acoustic wave generator, can be disposed in the liquid  110  or at any other suitable location, and is controlled to send sonic vibrations through the device  102  to provide a similar mechanical shaking function. 
     As can be appreciated by one skilled in the art, the nucleic acid strands each include different combinations of bases A, C, G and T, which each contain a particular magnitude and polarity of ionic charge. The charge gradient between drain and source regions  104  and  106 , or otherwise across the opening  118 , will thus cause the charged nucleic acid strands to traverse the opening  118 . Alternatively, another voltage source (not shown) can be used to create a difference in voltage potential between the opening  118  and the liquid. Also, a pressure differential can be applied across the opening  118  to control the flow of the DNA independent from the voltage applied between the drain and source  104  and  106 . 
     In addition, the Sequencing device  102  can attract the nucleic acid strands to the opening  118  by applying a positive voltage to the medium  110  relative to the voltage source. Furthermore, the nucleic acid strands in the medium  110  can be pushed in and out of the opening  118  and be analyzed multiple times by reversing the polarity across drain and source regions  104  and  106 , respectively. 
     As described in more detail below, the opening  118  is configured to have a diameter within the nanometer range, for example, within the range of about 1 nm to about 10 nm Therefore, only one DNA strand can pass through opening  118  at any given time. As a DNA strand passes through opening  118 , the sequence of bases induce image charges which form a channel  119  between the drain and source regions  104  and  106  that extends vertically along the walls of the device defining opening  118 . As a voltage is applied between the source  136  and drain  128  by means of the voltage source  112 , these image charges in the channel flow from source to drain, resulting in a current flow which can be detected by the current meter  114 . The current exists in the channel as long as the charge is present in the opening  118 , and thus the device current detected by the current meter  114  is much larger than the current associated with the moving charge. For example, a singly charged ion passing through the opening  118  in one microsecond accounts for an ion current of 0.16 pA and a device current of 160 nA. 
     Alternatively, the bases induce a charge variation in channel  119 , leading to a current variation as detected by current meter  114 . Any variation of the ion flow through the opening due to the presence of the DNA strand would also cause a variation to the image charge in the channel  119  and results in a current variation as detected by current meter  114 . That is, the device current measured by current meter  114  will diminish from, for example, 80 .mu.A to 4 .mu.A. as the DNA strand  111  passes through opening  118 . 
     Each different type of bases A, C, G, and T induces a current having a particular magnitude and waveform representative of the particular charge associated with its respective type of bases. In other words, an A type base will induce a current in a channel between the drain and source regions of the nucleic acid sequencing device  102  having a magnitude and waveform indicative of the A type base. Similarly, the C, T and G bases will each induce a current having a particular magnitude and waveform. 
     An example of a waveform of the detected current is shown in  FIG. 3 , which symbolically illustrates the shape, magnitude, and time resolution of the expected signals generated by the presence of the A, C, G and T bases. The magnitude of current is typically in the microampere (.mu.A) range, which is a multiplication factor of 10.sup.6 greater than the ion current flowing through the opening  118 , which is in the picoampere range. A calculation of the electrostatic potential of the individual bases shows the complementary distribution of charges that lead to the hydrogen bonding. For example, the T-A and C-G pairs have similar distributions when paired viewed from the outside, but, when unpaired, as would be the case when analyzing single-stranded DNA, the surfaces where the hydrogen bonding occurs are distinctive. The larger A and G bases are roughly complementary (positive and negative reversed) on the hydrogen bonding surface with similar behavior for the smaller T and C bases. 
     Accordingly, as the DNA strand passes through opening  118 , the sequence of bases in the strand can be detected and thus ascertained by interpreting the waveform and magnitude of the induced current detected by current meter  114 . The system  100  therefore enables DNA sequencing to be performed in a very accurate and efficient manner. 
     Since the velocity of the electrons in the channel  119  is much larger than the velocity of the ions passing through the opening, the drain current is also much larger than the ion current through the opening  118 . For an ion velocity of 1 cm/s and an electron velocity of 10.sup.6 cm/s, an amplification of 1 million can be obtained. 
     Also, the presence of a DNA molecule can be detected by monitoring the current I.sub.p through the opening  118 . That is, the current I.sub.p through the opening reduces from 80 pA to 4 pA when a DNA molecule passes through the opening. This corresponds to 25 electronic charges per microsecond as the molecule passes through the opening. 
     Measurement of the device current rather than the current through the opening has the following advantages. The device current is much larger and therefore easier to measure. The larger current allows an accurate measurement over a short time interval, thereby measuring the charge associated with a single DNA base located between the two n-type regions. In comparison, the measurement of the current through the opening has a limited bandwidth, limited by the shot-noise associated with the random movement of charge through the opening  118 . For example, measuring a 1 pA current with a bandwidth of 10 MHz yields an equivalent noise current of 3.2 pA. Also, the device current can be measured even if the liquids on both sides of the opening  118  are not electrically isolated. That is, as discussed above, the sequencing device  102  is immersed in a single container of liquid. Multiple sequencers  102  can thus be immersed in a single container of liquid to enable multiple current measurements to be performed in parallel. Furthermore, the nanometer-sized opening  118  can be replaced by any other structure or method which brings the DNA molecule in close proximity to the two n-type regions, as discussed in more detail below. 
     The preferred method of fabricating a nucleic acid sequencing device  102  will now be described with reference to  FIGS. 4-16 . As shown in  FIG. 4 , the fabrication process begins with a wafer  120 , such as a silicon-on-insulator (SOI) substrate comprising a silicon substrate  122 , a silicon dioxide (SiO.sub.2) layer  124 , and a thin layer of p-type silicon  126 . In this example, the silicon substrate  122  has a thickness within the range of about 300 .mu.m to about 600 .mu.m, the silicon dioxide layer  124  has a thickness within the range of about 200 to 6400 nm, and the p-type silicon layer  126  has a thickness of about 1 .mu.m or less (e.g., within a range of about 10 nm to about 1000 nm). 
     As shown in  FIG. 5 , a doped n-type region  128  is created in the p-type silicon layer  126  by ion implantation, and annealing or diffusion of an n-type dopant, such as arsenic, phosphorous or the like. As illustrated, the n-type region  128  is a shallow region which does not pass entirely through p-type silicon  126 . A deep n-type region  130  is also created in the p-type silicon  126  as illustrated in  FIG. 5 . The deep n-type region  130  passes all the way through the p-type silicon  126  to silicon dioxide  124  and is created by known methods, such as diffusion, or ion implantation and annealing of an n-type material which can be identical or similar to the n-type material used to create n-type region  128 . As further illustrated in  FIG. 5 , the silicon substrate  122  is etched along its (111) plane by known etching methods, such as etching in potassium hydroxide (KOH) or the like. The back of the substrate  122  can also be etched with a teflon jig. As illustrated, the etching process etches away a central portion of silicon substrate  122  down to the silicon dioxide  124  to create an opening  132  in the silicon substrate  122 . 
     As shown in  FIG. 6 , the portion of the silicon dioxide  124  exposed in opening  132  is etched away by conventional etching methods, such as etching in hydrofluoric acid, reactive etching or the like. Another shallow n-type region  124  is created in the area of the p-type silicon  126  exposed at opening  132  by known methods, such implantation or diffusion of an n-type material identical or similar to those used to create n-type regions  128  and  130 . 
     Opening  118  (see  FIGS. 1 and 2 ) is then formed through the n-type region  128 , p-type silicon  126  and bottom n-type region  134  as shown, for example, in  FIGS. 7 and 8  by reactive ion etching (RIE) using Freon 14 (CF.sub.4), optical lithography, electron-beam lithography or any other fine-line lithography, which results in an opening having a diameter of about 10 nm. As shown in  FIG. 9 , the diameter of the opening can be further decreased by oxidizing the silicon, thus forming a silicon dioxide layer  136  over the p-type silicon layer  126  and the walls forming opening  118 . This oxidation can be formed by thermal oxidation of the silicon in an oxygen atmosphere at 800-1000.degree. C., for example. As shown in detail in  FIGS. 11 and 12 , the resulting oxide has a volume larger than the silicon consumed during the oxidation process, which further narrows the diameter of opening  118 . It is desirable if the diameter of opening  118  can be as small as 1 nm. 
     Although for illustration purposes  FIGS. 1 ,  2  and  3 - 9  show opening  118  as being a cylindrically-shaped opening, it is preferable for opening  118  to have a funnel shape as shown, for example, in  FIGS. 13 and 14 . This funnel-shaped opening  118  is created by performing V-groove etching of the (100) p-type silicon layer  126  using potassium hydroxide (KOH), which results in V-shaped grooves formed along the (111) planes  138  of the p-type silicon  126 . The V-shaped or funnel-shaped opening, as shown explicitly in  FIG. 14 , facilitates movement of a DNA strand through opening  118 , and minimizes the possibility that the DNA strand will become balled up upon itself and thus have difficulty passing through opening  118 . Oxidation and V-groove etching can be combined to yield even smaller openings. Additionally, anodic oxidation can be used instead of thermal oxidation, as described above. Anodic oxidation has the additional advantage of allowing for monitoring of the opening size during oxidation so that the process can be stopped when the optimum opening size is achieved. 
     Specifically, the opening  118  should be small enough to allow only one molecule of the DNA strand  111  to pass through at one time. Electron-Beam lithography can yield an opening  118  as small as 10 nm, but even smaller openings are needed. Oxidation of the silicon and V-groove etching as described above can be used to further reduce the opening to the desired size of 1-2 nm Oxidation of silicon is known to yield silicon dioxide with a volume which is about twice that of the silicon consumed during the oxidation. Oxidation of a small opening  118  will result in a gradually reduced opening size, thereby providing the desired opening size V-groove etching of (100) oriented silicon using KOH results in V-grooves formed by (111) planes. KOH etching through a square SiO.sub.2 or Si.sub.3N.sub.4 mask results in a funnel shaped opening with a square cross-section. Etching through the thin silicon layer results in an opening  118  on the other side, which is considerable smaller in size. 
     Oxidation and V-groove etching can also be combined to yield even smaller openings  118 . Anodic oxidation can be used instead of thermal oxidation, which has the additional advantage of enabling the size of the opening  118  to be monitored during the oxidation and the oxidation can be stopped when the appropriate size of the opening  118  is obtained. 
     Turning now to  FIG. 15 , holes  140  are etched into the silicon dioxide  136  to expose n-type region  128  and n-type region  130 . Metal contacts  142  are then deposited onto silicon dioxide layer  136  and into holes  140  to contact the respective n-type regions  128  and  130 . An insulator  144  is then deposited over metal contacts  142  as shown in  FIG. 16 , thus resulting in device  102  as shown in  FIG. 1 . 
     As further shown in  FIG. 1 , a portion of insulator  144  can be removed so that leads  116  can be connected to the n-type regions  128  and  130 , which thus form the drain regions  104  and source  106 , respectively. An additional insulator  146  is deposited over insulator  144  to seal the openings through which leads  116  extend to contact n-type regions  128  and  130 . The completed device  102  can then be operated to perform the DNA sequencing as discussed above. 
     To identify the bases of the DNA molecule, it is desirable to measure a single electronic charge. If the sequencing device  102  is made to have a length and width of 0.1 by 0.1 .mu.m, and the thickness of the silicon dioxide layer is 0.1 .mu.m along the walls of the opening  118 , a capacitance of 0.35 fF, a voltage variation of 0.45 mV, a device transconductance of 1 mS and a current variation of 0.5 nA are realized. Accordingly, a sequencing device  102  having these dimensions and characteristics can be used to detect a single electronic charge. The sequencing device  102  can further be reduced in size to obtain a sufficient special resolution to distinguish between different nucleotides. The sequencing device  102  is preferably made smaller to have an improved charge sensing capability. For example, the width of the sequencing device can be 10 nm, the length can be 10 nm, and the opening  118  can have a diameter of 1 nm. 
     Additional embodiments of the device  102  can also be fabricated. For example,  FIG. 17  illustrates a top view of a nucleic acid sequencing device according to another embodiment of the present invention. In this embodiment, the steps described above with regard to  FIGS. 3 through 16  are performed to form the n-type regions which ultimately form the drain and source regions. However, in this embodiment, the n-type region  128  shown, for example, in  FIG. 5 , is formed as four separate n-type regions,  150  in a p-type silicon layer similar to p-type silicon layer  126  described above. A silicon dioxide layer  152  covers the p-type silicon layer into which n-type regions  150  have been created. Holes  156  are etched into silicon dioxide layer  152  so that metal contacts  158  that are deposited on silicon dioxide layer  152  can contact n-type regions  150 . By detecting current flowing between the four drain regions formed by n-type regions  150  and the source region (not shown), the spatial orientation of the bases on the DNA strand passing through opening  152  can be detected. 
       FIG. 18  is a cross section of a nucleic acid sequencing device  160  according to another embodiment of the present invention. Similar to nucleic acid sequencing device  102 ,  160  includes a silicon substrate  162 , a silicon dioxide layer  164 , an n-type region  166  implanted in p-type silicon  168 , and a second n-type region  170  implanted in p-type silicon  168 . Nucleic acid sequencing device  160  further has an opening  172  passing therethrough. The opening can be cylindrical, or can be a V-shaped or funnel-shaped opening as described above. A silicon dioxide layer  174  covers p-type silicon layer  168 , n-type region  170  and n-type region  166  as shown, and decreases the diameter of opening  172  in the manner described above. An opening is etched into silicon dioxide layer  172  to allow a lead  176  to be attached to n-type region  170 . Another lead  176  is also attached to an exposed portion of n-type region  166 , so that a voltage source  178  can apply a potential across the drain region  180  formed by n-type region  170  and source region  182  formed n-type region  166 . The nucleic acid sequencing device  160  can thus be used to detect the bases of a DNA strand  182  in a manner described above. 
       FIG. 19  illustrates a DNA sequencing system  186  according to another embodiment of the present invention. System  186  includes a multi-layer nucleic acid sequencing device  188  which, in this example, comprises three MOSFET-type devices stacked on top of each other. That is, device  188  includes a silicon substrate  190  similar to silicon substrate  122  described above. A silicon dioxide layer  192  is present on silicon substrate  190 . The device  188  further includes an n-type doped silicon region  194 , a p-type silicon dioxide region  196 , an n-type doped silicon region  198 , a p-type silicon dioxide region  200 , an n-type doped region silicon region  202 , a p-type silicon dioxide region  204  and an n-type doped silicon region  206 . Regions  194  through  206  are stacked on top of each other as shown explicitly in  FIG. 19 . However, as can be appreciated by one skilled in the art, the polarity of the layers can be reversed for this embodiment; and for any of the other embodiments discussed herein. That is, the device  188  can comprise a p-type doped silicon region  194 , an n-type silicon dioxide region  196 , a p-type doped silicon region  198 , and so on. 
     Additionally, a thin silicon dioxide layer  208  is formed over the layers as illustrated, and is also formed on the walls forming opening  210  to decrease the diameter of opening  210  in a manner described above with regard to opening  118 . Also, opening  210  can be cylindrically shaped, a V-shaped groove or a funnel-shaped groove as described above. Holes are formed in silicon dioxide layer  208  so that leads  212  can be attached to regions  194 ,  198 ,  202  and  206  to couple voltage source  214 ,  216  and  218  and current meters  220 ,  222  and  224  to device  188  as will now be described. Voltage sources  214 ,  216  and  218  and current meters  220 ,  222  and  224  are similar to voltage source  112  and current meter  114 , respectively, as described above. 
     Specifically, leads  212  couple voltage source  214  and current meter  220  in series to n-type doped silicon region  202  and n-type doped silicon region  206 . Therefore, voltage source  214  applies a voltage across regions  202  and  206  which are separated by p-type silicon dioxide region  204 . Leads  212  also couple voltage source  216  and current meter  222  to n-type doped silicon region  198  and n-type doped silicon region  202  as shown. Furthermore, leads  212  couple voltage source  218  and current meter  224  to n-type doped silicon region  194  and n-type doped silicon region  202  as shown. Accordingly, as can be appreciated from  FIG. 19 , n-type doped silicon region  198  and n-type doped silicon region  194  act as the drain and source regions, respectively, of one MOSFET, n-type doped silicon region  202  and n-type doped silicon region  198  act as drain and source regions, respectively, of a second MOSFET, and n-type doped silicon region  206  and n-type doped silicon region  202  act as drain and source regions, respectively, of a third MOSFET. These three MOSFET type devices can measure the current induced by the bases of a DNA strand passing through opening  210 , and thus take multiple measurements of these bases to improve accuracy. 
     It is also noted that a nucleic acid sequencing device above can be configured to sense the bases of a nucleic acid strand without it being necessary for the DNA strand to pass through an opening in the devices, as shown in  FIGS. 20 and 21 . That is, using the techniques described above, a nucleic acid sequencing device  226 , similar to nucleic acid sequencing device  102  shown in  FIG. 1 , can be fabricated having its drain and source regions proximate to a surface. It is noted that like components shown in  FIGS. 1 ,  20  and  21  are identified with like reference numbers. However, in place of an opening  118 , one or more grooves  228  can optionally be formed in the surface extending from the drain region to the source region. Alternatively, no grooves are formed in the surface, but rather, the detection area for detecting nucleic acid strands  111  is present between the drain and source regions. Techniques similar to those discussed above, such as the application of voltage potentials by means of voltage sources  117  and  121 , and creation of a pressure differential in the container  108  can be used to move the nucleic acid strands  111  in a horizontal direction along the surface of the device over the grooves  228 . The bases in the nucleic acid strands create an image charge channel  230  between the drain and source regions which allows current to flow between the drain and source regions. The current induced in the nucleic acid sequencing device by the bases can be measured in a manner similar to that described above. 
     Again, it is noted that the device  226  differs from the other embodiments represented in  FIGS. 1 ,  17  and  19  in that the channel  230  containing the image charge is horizontal rather than vertical. The structure no longer contains an opening  118  as in the device  102  shown in  FIGS. 1 ,  17  and  19 , but rather this embodiment contains a charge sensitive region just above channel  230 . Similar to  FIG. 1 , the external electrodes  113  and  115  are used to apply an electric field which steers the nucleic acid strands  111  towards or away from the charge sensitive region. That is, the motion of the nucleic acid strands  111  is controlled by applying a voltage to the external electrodes  113  and  115  relative to the voltage applied to the doped regions  130 . Additional electrodes (not shown) can be added to move the nucleic acid strands  111  perpendicular to the plane shown in  FIG. 20 . 
     The charge sensitive region of the device is located just above the channel  230  and between the two doped regions  130 . Identification of individual bases requires that the distance between the two doped regions is on the order of a single base and that the motion of the nucleic acid strand  111  is such that each base is successively placed above the charge sensitive region. This horizontal configuration enables more parallel as well as sequential analysis of the nucleic acid strands  111  and does not require the fabrication of a small opening. Additional surface processing, such as the formation of grooves  228  as discussed above that channel the nucleic acid strands  111  can be used to further enhance this approach. 
     The horizontal embodiment shown in  FIGS. 19 and 20  is also of interest to detect the presence of a large number of nucleic acid strands  111 . For instance, using an electrophoresis gel as the medium, one starts by placing nucleic acid strands  111  of different length between the electrodes  113  and  115 . A negative voltage is applied to the electrodes  113  and  115 , relative to the doped regions  130 . The nucleic acid strands  111  will then move towards the charge sensitive regions. The smaller strands will move faster and the larger strands will move slower. The smaller strands will therefore arrive first at the charge sensitive region followed by the larger ones. The charge accumulated in the charge sensitive region and therefore also the image charge in the channel  230  therefore increases “staircase-like” with time. This results in a staircase-like increase or decrease of the current measured by current meter  114 . 
     While this operation does not yield the identification of the individual bases of a single DNA/RNA strand, it does provide a measurement of the length of the strands equivalent to the one obtained by an electrophoresis measurement. The advantage over standard electrophoresis is that a real-time measurement of the position of the DNA/RNA strands is obtained. In addition, the dimensions can be reduced dramatically since micron-sized devices can readily be made, while standard electrophoresis uses mm if not cm-sized drift regions. This size reduction leads to faster measurements requiring less DNA/RNA strands, while also reducing the cost of a single charge sensing device. 
     It is further noted that multiple DNA sensors (e.g., sequencing devices  102 ) can be organized into a two-dimensional array  300  with electronic addressing and readout as shown in  FIG. 22 . The array consists of cells  302 , which contain the sequencing devices  102  connected on one side to ground and on the other side connected to the source, for example, of a transistor  304 , so that the drain-source current of a sequencing device  102  will flow into the source of its corresponding transistor  304 . The gates of the transistors  304  are connected to the word lines  306 , which in turn are connected to a decoder  308 . The drain of the transistor  304  in each cell  302  is connected to a sense line  310 . The sense lines  310  are connected to a series of sense amplifiers, shown as sense amplifier  312 . 
     The array  300  is operated by supplying an address to the decoder  308  from a controller, such as a microprocessor or the like (not shown). The decoder  308  then applies a voltage to the word-line  306  corresponding to the address. The sense amplifiers  312  provide the bias voltage to the selected row of sequencing devices  102 . The bias voltage causes the flow of DNA molecules through the opening  118  in the selected sequencing device. The selected sequencing device  102  provides to its corresponding transistor  304  a current which is proportional to the charge of the individual nucleotides in the manner described above. The sense amplifiers measure the current of each sequencing device  102  that is selected. The array  300  thus enables multiple simultaneous measurements, which increasing the sequencing rate as compared to as single sequencing device  102  and also providing redundancy and additional tolerance to defective sequencing devices  102 . 
     In addition, any of the DNA sequencers described above (e.g., sequencing device  102 ) can contain an alternative to the barrier (e.g., oxide layer  136 ) between the semiconductor channel (e.g., channel  119  in sequencing device  102  shown in  FIG. 1 ) and the medium containing the DNA molecules (e.g., liquid  110  shown in  FIG. 1 ). For example, the oxide barrier  136  can be removed, which still leaves a potential barrier between the semiconductor and the medium. The oxide layer  136  can be replaced by a wider band gap semiconductor doped with donors and/or acceptors. The oxide layer  136  can also be replaced by an undoped wider bandgap semiconductor layer. 
     Furthermore, the oxide layer  136  can be replaced with an oxide containing one or more silicon nanocrystals. The operation of a sequencing device  102  with this type of a barrier is somewhat different compared than that of a sequencing device  102  with an oxide layer  136 . That is, rather than directly creating an image charge in the semiconductor channel  119 , the charge of the individual nucleotides polarizes the nanocrystal in the barrier. This polarization of the nanocrystal creates an image charge in the semiconductor channel  119 . The sensitivity of the sequencing device  102  will be further enhanced as electrons tunnel from the nucleotide into the nanocrystal. The charge accumulated in the nanocrystal can be removed after the measurement (e.g., current reading by current meter  114 ) by applying a short voltage pulse across the drain and source of the sequencing device  102 . 
     Any of the sequencing devices described above (e.g., sequencing device  102 ) can also be constructed without the use of a semiconductor. In this arrangement, the middle p-type semiconductor  126  (see  FIG. 1 ) is replaced with an insulating layer such as silicon dioxide, while the n-type source region  106  and drain region  104  are replaced by metal electrodes. The oxide layer between the two metal electrodes must be thin enough (less than 10 nm) so that electrons can tunnel through the oxide layer. The oxide separating the two metal electrodes can be made thinner around the opening, so that tunneling only occurs at the opening. 
     The operation of this type of sequencing device  102 - 1  is described as follows with reference to  FIG. 23 . As a DNA molecule passes by the thin oxide layer, it changes the local potential in the oxide and causes a current variation due to tunneling of electrons through the oxide layer. Since the barrier separating the molecules from the channel  119  is very thin, tunneling of electrons can occur to and from the molecules. This tunneling can take place to/from the molecule from/to the channel  119 . This current is expected to be much smaller than the current in the channel  119  because of the large amplification within the sequencing device. However tunneling to and from the nanocrystal (as described above as an alternate barrier material) will provide a similar amplification. This tunneling will therefore provide useful and measurable information about the charge distribution along the DNA molecule. 
     As discussed above, the size of the opening in the sequencing device (e.g., opening  118  in sequencing device  102 ) can be varied over a large range. However for proper operation, the opening  118  must be small enough so that the DNA is in close proximity to the charge sensor and large enough so that the DNA can traverse the opening. Since the diameter of a single stranded DNA molecule equals about 1.5 nm, the opening should be between 1 and 3 nm for optimal sensing. Larger openings may result in reduced signal to noise ratio, but would provide a larger ion flow through the opening  118 . 
     It is further noted that certain advantages can be achieved by making the opening in the sequencing device asymmetric. For example, as shown in  FIGS. 24 and 25 , which correspond to  FIGS. 13 and 14  as their related discussion above, by making the initial pattern before V-groove etching asymmetric, the final opening  118  will also be asymmetric, for example, an oval or a rectangle. Then the nucleotides, which are asymmetric, will have a preferred orientation as they pass through the opening  118 . This removes the ambiguity in identifying the properties of the nucleotides due to their rotation around their backbone axis, and greatly simplifies analysis of the sensor signals. In addition, an electric field could be imposed along the longer axis of the opening  118  to align the base intrinsic dipole moment of the nucleotide with the field. For example, the dipole moment of Cytosine is 6.44 Debye, the dipole moment of Thymine is 4.50 Debye, the dipole moment of Adenine is 2.66 Debye and the dipole moment of Guanine is 6.88 Debye. If the field is strong enough, it can stretch the base (nucleotide) along the dipole moment, thus bringing the charges on the base nearer to the sensors increasing sensitivity. These techniques will thus make the data much easier to interpret, and will increase the signal used to discriminate between bases. 
     In addition, as shown in  FIG. 26 , array  300  of the sequencing devices described above (e.g. sequencing device  102 ) can be used in an arrangement in which the liquid in a vessel  400  is divided into two regions, namely, a source region  402  and one or more collection regions  404 . The source region  402  contains ions, fluid such as water, gel or the other types of liquids described above, and several kinds of nucleic acid strands  406 - 1  and  406 - 2 , such as DNA strands. 
     As shown, a collection region  404  can be formed by a collection vessel  408 . The collection vessel  408  can isolate the liquid in collection region  404  from the fluid in source region  402 , in which case the fluid in source region  402  can be the same as or different than the fluid in collection region  404 . Alternatively, the collection vessel  408  can be porous to allow the fluid in collection region  404  to flow into source region  402  and vice-versa while being impermeable to the nucleic acid strands  406 - 1  and  406 - 2 , thus prohibiting the nucleic acid strands  406 - 1  and  406 - 2  from passing through the collection vessel  408  from the source region  402  into the collection region  404  and vice-versa. 
     The source region might contain many extraneous DNA strands plus many strands of a particular type of DNA (type X) with a known or partially known nominal sequence. Then the array  300  can be controlled as described above to draw DNA strands through the openings (e.g., openings  118 ) in the N sequencing devices  102  operating in the array  300 , where N is a number much greater than 1 (e.g., 100 or more). As DNA strands traverse the openings  118  and are sequenced, the extraneous strands are backed out into the source region, which is manipulated so that this strand is removed from the vicinity of the opening  118  it just exited and has a negligible chance of entering another opening. 
     For example, strands of type X with the nominal sequence are counted, but also rejected and sent back into the source region. However, strands of type X with one or a few differences in the base sequence detected by nano-opening j (j=1 to N) are made to traverse the openings  118  and are captured in a container containing a collection region of the fluid. Any subsequent strands of type X identical to the first strand so captured are also counted and sent to an appropriate collection container. A different (or accidentally the same) non-nominal sequence of an X strand is collected at each opening  118 . When sequencing stops, the ratio of each type of non-nominal X strand to the nominal X strands is known, and a 100% pure sample of each variant type has been collected in the individual collection vessels  408 . These pure samples then can be duplicated by PCR and studied individually. This process can be used, for example, in studying mutation and/or incorrect duplication rates, and therefore aging, in DNA from an individual. 
     All of the devices described above can also be modified in other ways. For example, the SiO.sub.2 oxide layer can be converted to Si.sub.3N.sub.4 in a nitrous oxide (NO) ambient for use in alkaline solutions. Furthermore, since DNA molecules  111  are negatively charged, the molecules  111  can be attracted to the opening  118  by using electrodes, such as electrodes  113  and  115 , to apply a positive voltage to the liquid  110  relative to the source of the device. 
     As discussed above, a gel can be used in place of liquid  110  to contain the DNA molecules. The use of a gel will slow down the motion of the ions and further improve the signal to noise ratio. Furthermore, a pressure differential can be applied across the opening to control the flow independent from the applied voltage between source and drain. 
     Double stranded DNA can be analyzed as well. Even though double stranded DNA is a neutral molecule, since the molecule contains charge, the nucleotides can be identified by charge sensing. In addition, other molecules, for example, a fluorescent dye such as Hoechst dye, can be attached to single stranded DNA to enhance/modify the stiffness of the molecule thereby facilitating the insertion of the molecule into the nanometer-sized opening. Furthermore, since the above devices can by used to analyze generally any types of individual polymers, they can be used in industries dealing with polymers such as the petroleum industry, pharmaceutical industry and synthetic fiber industry, to name a few. 
     In addition, to facilitate the measurement of the charge of a single molecule (e.g., a nucleic acid strand  111  as shown in  FIG. 1 ), a larger size particle can be attached to a single molecule. For example, a gold nanoparticle can be attached to single-stranded DNA 6. The purpose of the gold nanoparticle is to provide a solid anchor to the DNA molecule. The gold particle can easily be charged and discharged. As a result, the gold particle and the attached DNA molecule can be manipulated by applying electric fields. 
     The gold nanoparticle attached to single stranded DNA will then be placed in an insulating liquid such as synthetic oil, which can be used as the liquid  110  in the arrangement shown in  FIG. 1 , for example. The purpose of the liquid is to allow the particle together with the DNA strand to move freely. The liquid should be insulating to avoid charge shielding by ions, which are present in conducting liquids. Synthetic oils have been identified as good candidates since they are highly resistive and do not form chemical bonds with single stranded DNA. 
     The charge of the gold nanoparticles can then be measured using a semiconductor based charge sensor, such as device  102  shown in  FIG. 1  that includes a floating gate, or device  226  without a gate electrode as shown in  FIG. 21 . As charged particles approach the device, image charges are formed at the silicon/oxide interface in the manner described above. An appropriate bias will be applied to the device (e.g., device  102 ) so that it operates in the sub threshold regime, where it is most sensitive to any image charge. The induced image charge then results in an increased conductivity of the device and is read out in the form of an increased current. As an example, it can be easily calculated that 100 DNA molecules, which each contain 100 bases that each carry about one third of an electronic charge, shift the threshold voltage of a 1 .mu.m by 1 .mu.m MOSFET with a 10 nm thick oxide by 150 mV. This shift can be measured by measuring the change in drain current of the device (e.g., as can be measured by current meter  114  shown in  FIG. 1 ). The measured charge is expected to be affected by the presence or absence of charge-shielding cations and stray ions in DNA&#39;s hydrogen shell. Also, electric fields can be used to separate the small ions from the larger DNA molecules. 
     It is further noted that the types of nanometer-size openings described above, for example, opening  118  in device  102  shown in  FIG. 1 , can be made as well-defined square holes as shown in  FIGS. 27A and 27B . To form these holes, a series of lines with the appropriate width and spacing is defined in a pattern, and the pattern is then transferred into a masking material such as SiO.sub.2. The same line pattern rotated by 90.degree. is then defined and transferred into the underlying masking material so that only the area defined by the overlapping areas between the two sets of lines is removed during etching. This process leads to a much better edge definition of the holes compared to defining the holes in a single lithographic step, as can be appreciated from the pattern  450  shown in  FIG. 27A  having openings  452 . The pattern shown in  FIGS. 27A and 27B  was made with the technique described above using a line pattern with 3 .mu.m width and 3 .mu.m spacing. The resulting etch mask was then used to etch the pits in the silicon using potassium hydroxide (KOH). 
     A further reduction of the line width can be achieved using electron-beam lithography. For example, electron-beam lithography using a Phillips 515 scanning electron microscope (SEM) can produce a line pattern with a 100 nm width. Polymethyl methacrylite (PMMA) can be used as an electron resist and developed with methyl iso butyl ketone/isopropyl alcohol (MIBK/IPA) to achieve the pattern  460  shown in  FIGS. 28A and 28B  having openings  462 . As illustrated, the lines are well defined and are limited by the spot size of the beam used in the electron-beam lithography. The beginning of each line is rounded since a single exposure with a gaussian beam has been used. This rounding can be eliminated by using the crossed line lithography technique described with regard to  FIGS. 27A and 27B . The PMMA can also be used as an etch mask to successfully transfer the pattern into a thin SiO.sub.2 layer as shown. 
     Accordingly, an opening  118  can be fabricated on (100) silicon membranes by combining state-of-the-art electron beam lithography with two well-known size reduction techniques discussed above. A scanning transmission electron microscope (STEM) can be used to define 10 nm lines in PMMA. Crossed lines will be used to create 10 nm square holes in a SiO.sub.2 mask. KOH etching can be used to etch V-shaped pits, providing a 2-4 nm opening on the other side of the silicon membrane. The opening will be further reduced in size by thermal oxidation of the silicon as it results in an oxide, which has about twice the volume of the oxidized silicon. This oxidation also provides the gate oxide, as discussed above. 
     Although only several exemplary embodiments of the present invention have been described in detail above, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the following claims.