JP4719906B2 - Field effect transistor device for ultrafast nucleic acid sequencing - Google Patents

Description

This application is based on US Pat. No. 119 (e), John R., named “Ultra-Fast, Semiconductor-Based Gene Sequencing”. US Patent Provisional Application No. 60 / 199,130 to Jon R. Sauer et al., Filed April 24, 2000, “Charge Sensing and Amplification Device for DNA” US Patent Provisional Application No. 60 / 217,681, filed July 12, 2000, and “Character Detection and Amplification for DNA Sequencing”, entitled “Sequencing”) ” John R., named “Charge Sensing and Amplification Device for DNA Sequencing”. US Patent Provisional Application No. 60 / 259,584 to Jon R. Sauer et al., Claiming the benefit of Jan. 4, 2001, filed under US Patent Law Section 120, “Ultrafast Nucleic Acid Sequences” John R., named “Determination device and method for making and using the same” (“An Ultra-Fast Nucleic Acid Sequencing Device and a Method for Making and Using the Same”). No. 09 / 653,543, filed on August 31, 2001, which is a non-provisional application of Jon R. Sauer et al. In the continuation-in-part application, the entire contents of both the provisional application and the non-provisional application are incorporated herein by reference.
Background of the Invention
Field of Invention
The present invention uses a semiconductor device having individual detection elements to identify individual bases of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and individual components (mers) of long-chain polymers such as carbohydrates and proteins. And a method for fabricating a semiconductor device. In particular, the present invention relates to a system and method using a semiconductor device similar to a field effect transistor device that can identify the base of a DNA / RNA strand and thereby enable sequencing of the strand of interest.
Explanation of related technology
DNA consists of two very long helical polynucleotide strands wound around a common axis. The two strands of the double helix are in opposite directions. The two chains are organized by hydrogen bonding between a pair 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. Therefore, one strand of the double helix is complementary to the other.
[0001]
Genetic information is encoded in the specific sequence of bases along the DNA strand. In normal cells, genetic information is passed from DNA to RNA. The majority of RNA molecules are single stranded, but most contain extensive double helix regions resulting from the strands breaking into a hairpin-like structure.
DNA sequence mapping is part of a new era of gene-based medicine embodied by the Human Genome Project. The effort of this plan will someday allow doctors to tailor treatment to the person based on the individual's genetic makeup, and may even be able to correct genetic defects before birth. However, to achieve this task, it is necessary to sequence each individual's DNA. Although the human genome sequence difference is approximately 0.1%, this small difference is important for understanding the predisposition to various illnesses in an individual. In the near future, medical care will be “DNA personalized” and doctors will be able to arrange sequence information as easily as they arrange today's cholesterol tests. Therefore, a faster and more economical DNA sequencing method is required in order to use such developments in everyday life.
[0002]
One method for performing DNA sequencing is disclosed in US Pat. No. 5,653,939, the entire contents of which are hereby incorporated by reference. This method uses a monolithic array of test sites formed on a substrate, such as a semiconductor substrate. Each test site includes a probe that is adapted to bind to a predetermined target molecular structure. Binding of the molecular structure to the probe at the test site changes the electrical, mechanical and optical properties of the test site. Thus, given a signal to the test site, the electrical, mechanical and optical properties of each test site can be measured to determine which probes have bound to their respective target molecular structures. However, this method is disadvantageous because test site arrays are complex to manufacture and must use multiple probes to detect various types of target molecular structures.
[0003]
Another method of sequencing is known as gel electrophoresis. In this technique, DNA is broken down into one strand and exposed to a chemical that destroys one of four nucleotides, eg, A, so that it ends with A and is labeled at the opposite end. Generate chains with a random distribution of fragments. Repeat for the other three remaining bases. DNA fragments are separated according to length by gel electrophoresis. The length indicates the distance from the labeled end to the known base. If there is no gap in the target range, the sequence of the original DNA strand fragment is determined.
[0004]
This DNA sequencing method has many disadvantages associated with it. This technique can only read about 500 bases. This is because a DNA strand containing more bases will be “rounded” and cannot be read correctly. Also, as the length of the chain increases, the resolution of length determination decreases rapidly, which also limits the analysis of the chain to 500 bases in length. In addition, gel electrophoresis is very time consuming and is not an effective solution to the problem of sequencing the genomes of complex organisms. Furthermore, pre-electrophoresis preparation and post-analysis are inherently expensive and time consuming. Therefore, there is a need for faster, consistent and more economical methods for DNA sequencing.
[0005]
Another approach to DNA sequencing is described in US Pat. Nos. 5,795,782 and 6,015,714, the entire contents of which are hereby incorporated by reference. In this technique, two liquids are separated by a biological membrane having alpha hemolysin pores. As DNA passes through the membrane, the ionic current through the pores is blocked. Experiments have shown that the length of time that the ionic current through the pore is blocked is proportional to the length of the DNA fragment. Furthermore, the amount and rate of blockage depends on which base is in the narrowest part of the pore. For this reason, there is a possibility that the base sequence can be determined from these phenomena.
[0006]
The problem with this technique is that so far individual nucleotides cannot be distinguished. Also, it is difficult to distinguish the spatial orientation of individual nucleotides. In addition, the electrode that measures the flow of charge is far away from the hole, which adversely affects the accuracy of the measurement. This is largely due to the specific capacitance of the current sensing electrode and the large statistical variation in the detection of small amounts of current. In addition, noise sources such as unique shot noise distort the signal, causing further errors. Therefore, there is a need for a more sensitive detection system that distinguishes bases as they pass through a sequencer.
Summary of the Invention
It is an object of the present invention to provide a system and method for accurately and effectively identifying individual bases of DNA or RNA.
[0007]
Another object of the present invention is to provide systems and methods that use semiconductor devices to sequence 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 individual components of long chain polymers such as carbohydrates or proteins and measuring the length of long chain polymers. It is.
[0008]
  Yet another object of the present invention is to have an opening therein for accurately and effectively identifying the base of DNA or RNA by measuring the charge at a position where the DNA or RNA molecule passes through the opening in the sequencer. Systems and methods using semiconductor-based devices are provided, thereby eliminating or at least minimizing the effects of noise sources such as shot noise associated with random movement of DNA or RNA molecules through openings. is there.
  These and other objects of the invention are:Determine the nucleic acid sequence of the nucleic acid strandThis is substantially achieved by providing a system for doing so. This system is close to the detection areaNucleic acid strandIncluding at least one semiconductor device having at least one detection region adapted to detect a charge representative of a component of the same or a structure of insulating and metal layers. Since the component can include the base of the nucleic acid chain, the detection region is adapted to detect a charge representing the base of the nucleic acid chain. The detection area is further adapted to generate a signal representative of the detected charge. Further, the detection region is a recess in the semiconductor device, orNucleic acid strandThe detection region can detect the charge of the component in the opening because the opening can include a region of the semiconductor device in which the opening is formed in a semiconductor device having a cross section sufficient to allow the opening to enter the opening . Furthermore, the semiconductor device preferably further comprises at least two doped regions, the detection region being able to send a current between the two doped regions in response to the presence of components near the detection region.
[0009]
  The above and other objects of the present invention provide for the proximity of the detection region of at least one semiconductor device.Nucleic acid strandPositioning a part of theNucleic acid strandDetecting a charge representative of a component ofDetermine the nucleic acid sequence of the nucleic acid strandThis is also substantially achieved by providing a method for doing so. Since the component can include the base of the nucleic acid strand, the detection step detects a charge representing the base. The method further includes generating a signal representative of the detected charge in the detection region. The detection region is a recess in the semiconductor device, orNucleic acid strandThe step of detecting can include a region of the semiconductor device in which the opening is formed in the semiconductor device having a cross-section sufficient to allow the opening to enter the opening. Is detected. Further, since the semiconductor device can further include at least two doped regions, the method can further include sending a current between the two doped regions in response to the presence of a component near the detection region.
[0010]
  These and other objects of the present invention include providing a semiconductor structure that includes at least one semiconductor layer, and a detection region near the detection region.Nucleic acid strandCreating a detection region in the semiconductor structure to detect charges representing components ofDetermine the nucleic acid sequence of the nucleic acid strandFurther substantially achieved by providing a method for manufacturing an apparatus for doing so. The component can include a base of the nucleic acid chain and a detection region can be created to detect a charge representing the base of the nucleic acid chain. The method creates a recess in the semiconductor structure that is positioned relative to the detection region such that the detection region detects charges representing a component in the recess or opening, orNucleic acid strandThe method further includes creating an opening in the semiconductor structure having a sufficient cross-section to allow a portion of the substrate to pass through. The method may further include forming an insulating layer on a wall surface of the semiconductor layer having the opening to reduce a cross section of the opening. In addition, the method provides for a detection area near the detection area.Nucleic acid strandCreating at least two doped regions in the semiconductor layer positioned with respect to the detection region so as to send current between the doped regions in response to the components of the semiconductor layer. The doped regions can be separated by portions of the semiconductor layer having different dopings, each having a first doping and can be created as a stack of doped regions separated by a layer having a second doping. . The doped region can include either p-type or n-type doping.
Brief Description of Drawings
  These and other objects, advantages and novel features of the present invention will be more readily understood from the following detailed description when read in conjunction with the accompanying drawings. In the drawing:
  FIG. 1 illustrates a system for performing DNA or RNA sequencing including a DNA or RNA sequencer configured in accordance with an embodiment of the present invention;
  FIG. 2 shows a plan view of the DNA or RNA sequencer shown in FIG. 1;
  FIG. 3 shows the current detector of the system shown in FIG. 1 as the adenine (A), thymine (T), guanine (G) and cytosine (C) bases of the DNA or RNA sequence pass through the DNA or RNA sequencer. FIG. 6 is a graph showing an example of a waveform representing a detected current;
  FIG. 4 shows a cross-sectional view of a silicon-on-insulator (SOI) substrate for making a DNA or RNA sequencer as shown in FIG. 1 according to an embodiment of the present invention;
  FIG. 5 shows a cross-sectional view of the SOI substrate shown in FIG. 4 with a shallow n-type region and a deep n-type region formed in the silicon layer and a portion of the substrate cut away;
  FIG. 6 shows a cross-sectional view of the SOI substrate shown in FIG. 5 with a portion of the insulator cut away and another shallow n-type region formed in the silicon layer;
  FIG. 7 shows a cross-sectional view of an SOI substrate having an opening formed by etching;
  FIG. 8 shows a plan view of the SOI substrate shown in FIG. 7;
  FIG. 9 shows a cross-sectional view of the SOI substrate shown in FIG. 7 with an oxide layer formed on the silicon layer and on the wall surface forming the opening therein;
  FIG. 10 shows a plan view of the SOI substrate shown in FIG. 9;
  FIG. 11 shows a detailed cross-sectional view of the SOI substrate shown in FIG. 9 with an oxide layer formed on the silicon layer and on the wall surface forming the opening therein;
  FIG. 12 shows a plan view of the SOI substrate shown in FIG. 11;
  FIG. 13 shows a detailed cross-sectional view of a configuration example of the opening of the SOI substrate shown in FIG. 7;
  FIG. 14 shows a plan view of the opening shown in FIG. 13;
  FIG. 15 shows a cross-sectional view of the SOI substrate shown in FIG. 9 with holes etched in the oxide layer and metal contacts formed respectively above the holes to contact the shallow and deep n-type regions;
  FIG. 16 shows a cross-sectional view of the DNA or RNA sequencer shown in FIG. 1 made according to the manufacturing steps shown in FIGS. 4-15;
  FIG. 17 shows a plan view of a DNA or RNA sequencer having a plurality of detectors formed by a plurality of n-type regions according to another embodiment of the invention;
  FIG. 18 shows a cross-sectional view of a DNA or RNA sequencer according to another embodiment of the invention;
  FIG. 19 shows a cross-sectional view of a DNA or RNA sequencer according to a further embodiment of the invention;
  FIG. 20 shows a cross-sectional view of a DNA or RNA sequencer according to a further embodiment of the invention;
  FIG. 21 shows a plan view of the DNA or RNA sequencer shown in FIG.
[0011]
FIG. 22 is a conceptual block diagram illustrating an example of a matrix arrangement of a DNA or RNA sequencer;
FIG. 23 is a cross-sectional view of a sequencer having a metal layer instead of an intermediate semiconductor layer to achieve electron tunneling;
24 shows a detailed cross-sectional view of another configuration example of the opening of the SOI substrate shown in FIG. 7;
FIG. 25 shows a plan view of the opening shown in FIG. 24;
FIG. 26 shows a cross-sectional view of a multi-opening sequencer used with a separate liquid region;
Figures 27A and 27B are photographic images of opening patterns formed in a semiconductor structure;
28A and 28B are photographic images of opening patterns formed in a semiconductor structure.
Detailed Description of the Preferred Embodiment
FIGS. 1 and 2 show individual components and polymers or polymers or polymers for detecting the presence of a polymer such as DNA or RNA, protein or carbohydrate, or a long chain polymer such as petroleum, more preferably 1 illustrates a system 100 for identifying the length of a long chain polymer. The system 100 is preferably adapted for sequencing nucleic acids such as DNA or RNA sequencing according to embodiments of the present invention. Accordingly, the system 100 is described herein in connection with nucleic acid sequencing.
[0012]
The system 100 includes a nucleic acid sequencing device 102 that is a semiconductor device, as will be described in more detail below. In particular, the nucleic acid sequencing device 102 is similar to 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 MOSFETs, nucleic acid sequencing devices do not include a gate region for the reasons described below.
The nucleic acid sequencing device 102 is placed in a container 108 containing a liquid 110, such as water, a gel, a buffer such as KCL, or any other suitable solution. Note that the solution 110 may be an insulating medium such as oil or any other suitable insulating medium. Further, the container 108 may not include a medium such as a liquid. That is, the container 108 can be sealed and evacuated to create a vacuum in which the nucleic acid sequencing device 102 is placed. Also, FIG. 1 shows only one nucleic acid sequencing device 102 in the container 108 as an example, but the container includes a plurality of nucleic acid sequencing devices 102 for performing a plurality of DNA sequencing measurements in parallel. be able to.
[0013]
The medium or vacuum, such as the liquid 110 in the container 108, contains a nucleic acid strand or portion 111 of the nucleic acid strand that is sequenced by the nucleic acid sequencing device 102. As further shown, a voltage source 112, such as a DC voltage source, is coupled in series with an ammeter 114 by a lead 116 through drain and source regions 104 and 106, respectively. In this example, the positive lead of voltage source 112 is coupled to drain region 104 and the negative lead of voltage source 112 is coupled to source region 106 via ammeter 114.
[0014]
The potential applied to the drain and source regions 104 and 106 of the nucleic acid sequencing device 102 may be as low as, for example, about 100 mV, which is used to draw the nucleic acid strand into the opening 118 of the nucleic acid sequencing device 102. It is sufficient to create a gradient in the source regions 104 and 106. That is, the nucleic acid strand 111 moves through the opening 118 with a local gradient. Alternatively or additionally, the liquid may comprise an ionic solution. In this case, the local gradient causes ions in the solution to flow through the opening 118, which helps the nucleic acid strand 111, such as DNA or RNA, to move through the opening 118 as well.
[0015]
Additional electrodes 113 and 115 placed in the medium 110 and connected to additional voltage sources 117 and 121 further facilitate movement of the nucleic acid strand toward the opening 118. That is, the external electrodes 113 and 115 are used for applying an electric field in the medium 110. This electric field causes all charged particles such as nucleic acid strand 111 to flow either towards opening 118 or away from opening 118. For this reason, the electrodes 113 and 115 are used as means for directing the nucleic acid strand 111 into or out of the opening 118. In order to connect the voltage sources 112 and 117 to the nucleic acid sequencer 102, metal contacts 123 are coupled to n-type doped regions 128 and 130, described in more detail below. The electrodes 113 and 115 may also be able to provide a high frequency voltage that is superimposed on the DC voltage by the AC voltage source 125. This high frequency voltage, which can have a frequency in the radio frequency range such as the megahertz range (eg 10 MHz), causes the nucleic acid strand 111 and ions to vibrate. This vibration allows the nucleic acid strand 111 to pass more smoothly through the opening 118 in a manner similar to shaking the salt shaker so that salt particles can pass through the opening of the shaker. Alternatively, a device 127, such as a sonic generator, can be placed in the liquid 110 or at any other suitable location, and sonic vibrations can be transmitted through the device 102 to provide a similar mechanical shaking function. Controlled to send.
[0016]
As will be appreciated by those skilled in the art, nucleic acid strands each contain different combinations of bases A, C, G and T, each containing an ionic charge of a particular size and polarity. Thus, a charge gradient between the drain and source regions 104 and 106 or otherwise across the opening 118 causes the charged nucleic acid strand to pass through the opening 118. Alternatively, another voltage source (not shown) can be used to create a potential difference between the opening 118 and the liquid. In addition, a pressure differential can be applied across the opening 118 to control the flow of DNA independent of the voltage applied between the source and drain 106 and 104.
[0017]
In addition, the sequencing apparatus 102 can attract the nucleic acid chain to the opening 118 by applying a positive voltage to the medium 110 with the voltage source 112 as a reference. Furthermore, by reversing the polarities of the drain and source regions 104 and 106, the nucleic acid strand in the medium 110 can be pushed into and pushed out of the opening 118 and analyzed multiple times.
As described in more detail below, the opening 118 is configured to have a diameter in the nanometer range, such as in the range of about 1 nm to about 10 nm. Therefore, only one DNA strand can always pass through the opening 118. As the DNA strand passes through the opening 118, the sequence of bases induces a mirror image charge. This mirror image charge forms a channel 119 between the drain and source regions 104 and 106 that extends vertically along the wall of the device in which the opening 118 is formed. When a voltage is applied between source region 106 and drain region 104 by voltage source 112, these mirror image charges in the channel flow from source to drain, resulting in a current flow that can be detected by ammeter 114. . The device current detected by ammeter 114 is much larger than the current associated with the mobile charge because current is present in the channel as long as there is charge in opening 118. For example, every single charged ion that passes through the aperture 118 in 1 microsecond causes an ion current of 0.16 pA and a device current of 160 nA.
[0018]
Alternatively, the base induces a charge change in channel 119 resulting in a change in current as detected by ammeter 114. Changes in the flow of ions through the opening due to the presence of DNA strands also cause a change in the image charge in channel 119, resulting in a change in current detected by ammeter 114. That is, the device current measured by the ammeter 114 decreases from, for example, 80 μA to 4 μA when the DNA strand 111 passes through the opening 118.
Each type of base A, C, G and T induces a current having a specific magnitude and waveform representing a specific charge associated with the type of the respective base. That is, the A type base induces a current having a magnitude and a waveform indicating that it is an A type base in the channel between the drain and source regions of the nucleic acid sequencing device 102. Similarly, C, T and G bases induce currents with specific magnitudes and waveforms, respectively.
[0019]
An example of the detected current waveform is shown in FIG. This figure symbolically illustrates the shape, magnitude and temporal resolution of the expected signal produced by the presence of A, C, G and T bases. The magnitude of the current is typically in the microampere (μA) range, which is 10 times the ion current flowing through the opening 118 in the picoampere range.⁶Double the size. Calculation of the electrostatic potential of the individual bases shows a complementary distribution of charges resulting in hydrogen bonding. For example, the TA and CG pairs have a similar distribution when viewed in pairs, but when analyzing non-paired single-stranded DNA, the surface where hydrogen bonding occurs is unique. Large A and G bases are nearly complementary (positive and negative are reversed) on the hydrogen bonding surface, as are small T and C bases.
[0020]
Thus, as the DNA strand passes through the opening 118, the base sequence of the strand can be detected and therefore confirmed by interpreting the waveform and magnitude of the induced current detected by the ammeter 114. Thus, the system 100 allows DNA sequencing to be performed in a very accurate and efficient manner.
Since the velocity of electrons in channel 119 is much greater than the velocity of ions passing through the aperture, the drain current is also much greater than the ion current through aperture 118. Ion velocity 1 cm / sec and electron velocity 10⁶In the case of cm / second, amplification of 1 million times is obtained.
[0021]
In addition, the presence of DNA molecules is caused by the current I through the opening 118._PCan be detected by monitoring. That is, the current I through the opening I_PDecreases from 80 pA to 4 pA as DNA molecules pass through the opening. This corresponds to a charge of 25 electrons per microsecond as the molecule passes through the opening.
Measuring device current rather than current through the aperture has the following advantages. The device current is relatively large and easy to measure. A large current allows an accurate measurement in a short time, thereby allowing the measurement of the charge associated with one DNA base located between two n-type regions. In contrast, the measurement bandwidth of current through the aperture is limited by shot noise associated with random movement of charge through the aperture 118. For example, when a current of 1 pA is measured with a bandwidth of 10 MHz, a noise current corresponding to 3.2 pA is generated. Further, the device current can be measured even if the liquid on both sides of the opening 118 is not electrically insulated. That is, as described above, the sequencing apparatus 102 is immersed in a single liquid container. Therefore, a plurality of sequencers 102 can be immersed in a single liquid container so that a plurality of current measurements can be performed in parallel. Further, as described in more detail below, any other structure or method that allows the DNA molecule to be in close proximity to the two n-type regions in place of the nanometer-sized opening 118 can be used.
[0022]
A suitable method for producing the nucleic acid sequencing device 102 will be described with reference to FIGS. As shown in FIG. 4, the manufacturing process includes a silicon substrate 122, silicon dioxide (SiO 2).₂) Layer 124 and a thin p-type silicon layer 126, starting from a wafer 120 such as a silicon on insulator (SOI) substrate. In this example, silicon substrate 122 has a thickness in the range of about 300 μm to about 600 μm, silicon dioxide layer 124 has a thickness in the range of about 200 to 6400 nm, and p-type silicon layer 126 has a thickness of about 1 μm. It has a thickness of the following (for example, in the range of about 100 nm to about 1000 nm).
[0023]
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 n-type dopants such as arsenic, phosphorus, and the like. As illustrated, the n-type region 128 is a shallow region that does not completely penetrate the p-type silicon 126. A deep n-type region 130 is also created in the p-type silicon 126 as shown in FIG. Deep n-type region 130 penetrates p-type silicon 126 to silicon dioxide 124, diffusion of n-type material, or ion implantation, which may be the same as or similar to the n-type material used to create n-type region 128. And by known methods such as annealing. As further shown in FIG. 5, the silicon substrate 122 is etched along its (111) plane by known methods such as etching in potassium hydroxide (KOH). The back surface of the substrate 122 can also be etched with a teflon jig. As shown in the drawing, the etching process cuts the central portion of the silicon substrate 122 up to the silicon dioxide 124 to form an opening 132 in the silicon substrate 122.
[0024]
As shown in FIG. 6, the portion of the silicon dioxide 124 exposed in the opening 132 is cut away by a conventional etching method such as etching in hydrofluoric acid, reactive etching, or the like. Another shallow n is exposed to the location of the p-type silicon 126 exposed at the opening 132 by known methods such as implantation or diffusion of the same or similar n-type material used to create the n-type regions 128 and 130. A mold region 134 is created.
An opening 118 (see FIGS. 1 and 2) is then formed, for example as shown in FIGS. 7 and 8, with Freon 14 (CF_Four) Through reactive ion etching (RIE), optical lithography, electron beam lithography or other fine line lithography through the n-type region 128, the p-type silicon 126 and the bottom n-type region 134, with a diameter of about 10 nm. It becomes the opening part which has. As shown in FIG. 9, the diameter of the opening can be further reduced by forming a silicon dioxide layer 136 on the wall surface that forms the p-type silicon layer 126 and the opening 118 by oxidizing silicon. . This oxidation state can be formed, for example, by thermal oxidation of silicon in an oxygen atmosphere at 800 to 1000 ° C. As shown in detail in FIGS. 11 and 12, the resulting oxide has a larger volume than the silicon consumed during the oxidation process, which further narrows the diameter of the opening 118. It is desirable that the diameter of the opening 118 is as small as 1 nm.
[0025]
For purposes of illustration, FIGS. 1, 2 and 3-9 show the opening 118 as a cylindrical opening, but the opening 118 may have a funnel shape, for example as shown in FIGS. preferable. This funnel-shaped opening 118 is created by performing a V-shaped groove etch of the (100) p-type silicon layer 126 using potassium hydroxide (KOH), along the (111) plane 138 of the p-type silicon 126. It becomes a V-shaped groove formed. As clearly shown in FIG. 14, the V-shaped or funnel-shaped opening facilitates movement of the DNA strand through the opening 118 and makes it difficult to pass through the opening 118 because the DNA strand is rounded. Minimize the possibility. Oxidation and V-groove etching can be combined to produce even smaller openings. Further, as described above, anodic oxidation can be used instead of thermal oxidation. Anodization has the further advantage of allowing the opening size to be monitored during oxidation so that the process can be stopped once the optimum opening size is obtained.
[0026]
In particular, the opening 118 must be small enough to pass only one molecule of DNA strand at a time. In electron beam lithography, an opening 118 having a size as small as 10 nm can be formed, but a smaller opening is required. Silicon oxidation and V-groove etching as described above can be used to further reduce the opening to the desired size of 1-2 nm. It is known that silicon oxidation results in silicon dioxide having a volume approximately twice that of the silicon consumed during oxidation. Oxidation of the small openings 118 gradually reduces the opening size, thereby providing the desired opening size. V-groove etching of (100) oriented silicon using KOH results in a V-shaped groove formed by the (111) plane. Square SiO₂Or Si_ThreeN_FourKOH etching through the mask gives a funnel shaped opening with a square cross section. Etching a thin silicon layer results in a very small size opening 118 on the other side.
[0027]
A smaller opening 118 can also be formed by a combination of oxidation and V-groove etching. Instead of thermal oxidation, anodization can be used which has the additional advantage of allowing the size of the opening 118 to be monitored during oxidation, stopping the oxidation once an appropriately sized opening 118 is obtained. Can do.
Referring now to FIG. 15, a hole 140 is etched in the silicon dioxide 136 to expose the n-type region 128 and the n-type region 130. A metal contact 142 is then deposited on the silicon dioxide layer 136 and in the hole 140 to contact each n-type region 128 and 130. After that, an insulator 144 is deposited on the metal contact 142 as shown in FIG. 16, and thus the device 102 shown in FIG. 1 is obtained.
[0028]
As further shown in FIG. 1, a portion of the insulator 144 can be removed so that the lead 116 can contact the n-type regions 128 and 130, so that these regions are drain region 104 and source region 106, respectively. Form. Additional insulator 146 is deposited over insulator 144 to seal the opening through which lead 116 extends to contact n-type regions 128 and 130. As a result, the completed device 102 can be operated to perform DNA sequencing as described above.
[0029]
In order to identify the base of a DNA molecule, it is desirable to measure the charge of a single electron. If the sequencing device 102 is fabricated to have a length and width of 0.1 × 0.1 μm and the thickness of the silicon dioxide layer is 0.1 μm along the wall of the opening 118, then 0.35 fF Capacitance, 0.45 mV voltage change, 1 mS device transconductance and 0.5 nA current change. Thus, a sequencing device 102 having these dimensions and characteristics can be used to detect the charge of a single electron. The sequencing device 102 can be further reduced in size to obtain special resolution sufficient to distinguish different nucleotides. The sequencing device 102 is preferably smaller to have a higher charge sensing capability. For example, the sequencing device may have a width of 10 nm, a length of 10 nm, and the opening 118 may have a diameter of 1 nm.
[0030]
Further embodiments of the device 102 can also be made. For example, FIG. 17 shows a plan view of a nucleic acid sequencing device according to another embodiment of the present invention. In this embodiment, the steps described above with respect to FIGS. 3-16 are performed to form n-type regions that ultimately form drain and source regions. However, in this embodiment, for example, the n-type region 128 shown in FIG. 5 is formed in a p-type silicon layer similar to the p-type silicon layer 126 as four separate n-type regions 150. Silicon dioxide layer 154 covers the p-type silicon layer with n-type region 150 formed therein. The hole 156 is etched in the silicon dioxide layer 154 so that the metal contact 158 deposited on the silicon dioxide layer 154 can contact the n-type region 150. It is possible to detect the spatial orientation of the base of the DNA strand passing through the opening 152 by detecting the current flowing between the four drain regions and the source region (not shown) formed by the n-type region 150. it can.
[0031]
FIG. 18 is a cross section of a nucleic acid sequencing device 160 according to another embodiment of the invention. Similar to the nucleic acid sequencing device 102, the nucleic acid sequencing device 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 implanted in p-type silicon 168. A mold area 170 is included. The nucleic acid sequencing device 160 further has an opening 172 extending therethrough. The opening may be cylindrical or may be a V-shaped or funnel-shaped opening as described above. The silicon dioxide layer 174 covers the p-type silicon layer 168, the n-type region 170, and the n-type region 166 as shown, and reduces the diameter of the opening 172 as described above. An opening is etched in the silicon dioxide layer 174 to bond the lead 176 to the n-type region 170. Another lead 176 is also joined to the exposed portion of n-type region 166 so that voltage source 178 is between drain region 180 formed by n-type region 170 and source region 182 formed by n-type region 166. A potential can be applied to the capacitor. In this way, the nucleic acid sequencing device 160 can be used to detect the base of the DNA strand 184 in the manner described above.
[0032]
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 includes three MOSFET type devices stacked on top of each other. That is, the device 188 includes a silicon substrate 190 similar to the silicon substrate 122 described above. A silicon dioxide layer 192 is on the silicon substrate 190. Device 188 includes n-type doped silicon region 194, p-type silicon dioxide region 196, n-type doped silicon region 198, p-type silicon dioxide region 200, n-type doped silicon region 202, p-type silicon dioxide region 204 and n-type doped silicon. A region 206 is further included. Regions 194 to 206 are stacked on top of each other as clearly shown in FIG. However, the polarity of the layers can be reversed for this embodiment and all other embodiments described herein, as will be appreciated by those skilled in the art. That is, the device 188 may include a p-type doped silicon region 194, an n-type silicon dioxide region 196, a p-type doped silicon region 198, and the like.
[0033]
In addition, a thin silicon dioxide layer 208 is formed on the layer as shown, and is also formed on the wall forming the opening 210 to reduce the diameter of the opening 210 in the manner described above with respect to the opening 118. . In addition, the opening 210 may be a cylindrical shape, a V-shaped groove or a funnel-shaped groove as described above. The silicon dioxide layer so that leads 212 can be joined to regions 194, 198, 202 and 206 to couple voltage sources 214, 216 and 218 and ammeters 220, 222 and 224 to device 188 as described below. A hole is formed in 208. As described above, voltage sources 214, 216 and 218 and ammeters 220, 222 and 224 are similar to voltage source 112 and ammeter 114, respectively.
[0034]
In particular, lead 212 couples voltage source 214 and ammeter 220 in series with n-type doped silicon region 202 and n-type doped silicon region 206. Thus, voltage source 214 applies a voltage between regions 202 and 206 separated by p-type silicon dioxide region 204. Lead 212 also couples voltage source 216 and ammeter 222 to n-type doped silicon region 198 and n-type doped silicon region 202 as shown. In addition, lead 212 couples voltage source 218 and ammeter 224 to n-type doped silicon region 194 and n-type doped silicon region 202 as shown. As a result, as can be fully understood from FIG. 19, the n-type doped silicon region 198 and the n-type doped silicon region 194 act as the drain and source regions of the first MOSFET, respectively. Doped silicon region 198 acts as the drain and source regions of the second MOSFET, respectively, and n-type doped silicon region 206 and n-type doped silicon region 202 act as the drain and source regions of the third MOSFET, respectively. These three MOSFET type devices can measure the current induced by the bases of the DNA strand passing through the opening 210, thereby allowing multiple measurements of these bases to increase accuracy. it can.
[0035]
Also, as shown in FIGS. 20 and 21, the nucleic acid sequencing device described above can be configured to detect the base of the nucleic acid strand without requiring the DNA strand to pass through the opening of the device. . That is, using the above technique, a nucleic acid sequencing device 226 having a drain and a source region close to the surface, similar to the nucleic acid sequencing device 102 shown in FIG. 1, can be produced. Similar components shown in FIGS. 1, 20 and 21 are identified by similar reference numbers. However, instead of the 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 groove is formed on the surface, but the detection region for detecting the nucleic acid strand 111 is between the drain and source regions. Techniques similar to those described above, such as the application of a potential by voltage sources 117 and 121 and the generation of a pressure differential in vessel 108, move nucleic acid strand 111 horizontally over groove 228 along the surface of the device. Can be used. The base of the nucleic acid strand creates a mirror image charge channel 230 between the drain and source regions that allows current to flow between the drain and source regions. The current induced in the nucleic acid sequencing apparatus by the base can be measured by the same method as described above.
[0036]
Furthermore, the device 226 differs from the other embodiments depicted in FIGS. 1, 17 and 19 in that the channel 230 containing the mirror image charge is horizontal rather than vertical. Although this structure does not include the opening 118 as in the device 102 shown in FIGS. 1, 17 and 19, this embodiment includes a charge sensing region just above the channel 230. As in FIG. 1, external electrodes 113 and 115 are used to provide an electric field that directs the nucleic acid strand 111 toward or away from the charge sensing region. That is, the movement of the nucleic acid strand 111 is controlled by applying a voltage to the external electrodes 113 and 115 with the voltage applied to the doped region 130 as a reference. Additional electrodes (not shown) can also be added to move the nucleic acid strand 111 perpendicular to the plane shown in FIG.
[0037]
The charge sensing region of the device is located just above the channel 230 and between the two doped regions 130. For identification of individual bases, the distance between the two doped regions is about one base, and the nucleic acid strand 111 needs to move so that each base is continuously above the charge detection region. This horizontal configuration allows further parallel and continuous analysis of nucleic acid strands 111 and does not require the creation of small openings. To further enhance this approach, additional surface treatments such as the formation of grooves 228 as described above leading to the nucleic acid strand 111 can be used.
[0038]
The horizontal embodiment shown in FIGS. 19 and 20 is also advantageous for detecting the presence of multiple nucleic acid strands 111. For example, we begin by placing nucleic acid strands 111 of various lengths between electrodes 113 and 115 using an electrophoresis gel as a medium. A negative voltage is applied to electrodes 113 and 115 with reference to doped region 130. Then, the nucleic acid chain 111 moves toward the charge detection region. Small chains move fast and large chains move slowly. Thus, a small chain first arrives at the charge sensing region, followed by a larger one. Thus, the charge accumulated in the charge sensing region, and hence the mirror image charge of channel 230, also increases “stepwise” over time. As a result, the current measured by the ammeter 114 increases or decreases stepwise.
[0039]
This treatment does not identify individual bases of a single DNA / RNA strand, but provides a measure of strand length equivalent to that performed by electrophoretic measurements. The advantage over standard electrophoresis is that a real-time measurement of the DNA / RNA strand position is made. Also, even if the standard electrophoresis is not the cm size, the mm size migration region is used, whereas the micron size device can be easily manufactured, so that the size can be greatly reduced. This reduction in size results in faster measurements with fewer DNA / RNA strands required, while also reducing the cost of a single charge sensing device.
[0040]
In addition, a number of DNA sensors (eg, sequencing device 102) can be configured in a two-dimensional array 300 that is electronically addressed and read as shown in FIG. The array is from a cell 302 that includes a sequencing device 102 that is connected to ground on the one hand and connected to, for example, the source of transistor 304 so that the drain-source current of the sequencing device 102 flows to the source of the corresponding transistor 304. Become. The gate of the transistor 304 is connected to the word line 306, and the word line 306 is connected to the decoder 308. The drain of the transistor 304 of each cell 302 is connected to the sense line 310. The sense line 310 is connected to a series of sense amplifiers illustrated as sense amplifiers 312.
[0041]
The array 300 is operated by supplying addresses to a decoder 308 from a controller (not shown) such as a microprocessor. Then, the decoder 308 applies a voltage to the word line 306 corresponding to the address. The sense amplifier 312 provides a bias voltage to a selected column of sequencing devices 102. The bias voltage provides a flow of DNA molecules through the opening 118 in the selected sequencing device. The selected sequencing device 102 provides the corresponding transistor 304 with a current proportional to the charge of the individual nucleotides as described above. The sense amplifier measures the current of each selected sequencing device 102. In this way, the array 300 allows multiple simultaneous measurements to increase sequencing speed compared to a single sequencing device 102 and at the same time provide redundancy and additional tolerances for a defective sequencing device 102. enable.
[0042]
In addition, any of the above DNA sequencers (eg, sequencing device 102) may include a semiconductor channel (eg, channel 119 of sequencing device 102 shown in FIG. 1) and a medium containing DNA molecules (eg, the liquid shown in FIG. 1). 110) to the barrier (eg, oxide layer 136). For example, the oxide layer 136 can be removed and still leave a potential barrier between the semiconductor and the medium. Instead of the oxide layer 136, a semiconductor having a wide band gap doped with a donor and / or an acceptor can be used. Instead of the oxide layer 136, an undoped semiconductor layer having a wide band gap can be used.
[0043]
Further, an oxide including one or more silicon nanocrystals can be used instead of the oxide layer 136. The operation of sequencing device 102 with this type of barrier is slightly different from that of sequencing device 102 with oxide layer 136. That is, rather than creating a mirror image charge directly in the semiconductor channel 119, the charge of individual nucleotides polarizes the barrier nanocrystal. This polarization of the nanocrystal creates a mirror image charge in the semiconductor channel 119. Since the electrons tunnel from the nucleotide to the nanocrystal, the sensitivity of the sequencing device 102 is further enhanced. The charge accumulated in the nanocrystal can be removed after measurement (eg, reading the current with an ammeter 114) by applying a short voltage pulse across the drain and source of the sequencing device 102.
[0044]
Any of the above-described sequencing apparatuses (for example, the sequencing apparatus 102) can be constructed without using a semiconductor. In this configuration, an insulating layer such as silicon dioxide is used instead of the intermediate p-type semiconductor 126 (see FIG. 1), and a metal electrode is used instead of the n-type source region 106 and drain region 104. The oxide layer between the two metal electrodes must be thin enough (less than 10 nm) that electrons can tunnel through the oxide layer. The oxide separating the two metal electrodes may be thinner around the opening so that tunneling occurs only at the opening.
[0045]
The operation of this type of sequencing apparatus 102-1 will now be described with reference to FIG. As DNA molecules pass by a thin oxide layer, they change the local potential of the oxide, resulting in a change in current due to electron tunneling through the oxide layer. The barrier separating the molecule from the channel 119 is so thin that tunneling of electrons into and out of the molecule can occur. This tunneling can occur from channel 119 to molecule / from molecule to channel 119. This current is believed to be much smaller than the current in channel 119 due to large amplification in the sequencing device. However, tunneling from and into the nanocrystal (as described above as an alternative barrier material) results in similar amplification. This tunneling thus provides useful and measurable information about the distribution of charges along the DNA molecule.
[0046]
As described above, the size of the opening of the sequencing device (eg, opening 118 of sequencing device 102) can vary over a wide range. However, for proper operation, the aperture 118 must be small enough so that the DNA is very close to the charge sensor and large enough so that the DNA can pass through the aperture. Since the diameter of single stranded DNA is equal to about 1.5 nm, the opening should be between 1 nm and 3 nm for optimal detection. Larger openings may reduce the signal to noise (S / N) ratio, but larger ions will pass through the openings 118.
[0047]
In addition, several advantages are achieved by making the openings of the sequencing apparatus asymmetric. For example, as shown in FIGS. 24 and 25, which correspond to FIGS. 13 and 14 as the related description above, by making the initial pattern prior to V-groove etching asymmetric, the final opening 118 is also Asymmetric, for example, not an square or a circle, but an oval or a rectangle. Thus, nucleotides that are asymmetric have a preferred orientation when passing through opening 118. This removes the ambiguity due to rotation around their skeletal axes in identifying the characteristics of the nucleotides and greatly simplifies the analysis of sensor signals. Furthermore, an electric field can be applied along the long axis of the opening 118 to align the dipole moment inherent to the nucleotide base with the electric 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 electric field is sufficiently strong, the base (nucleotide) can be extended along the dipole moment, whereby the charge of the base can be brought closer to the sensor to increase the sensitivity. Therefore, these techniques make the data much easier to interpret and increase the signal used to determine the base.
[0048]
In addition, as shown in FIG. 26, the array 300 of the sequencing device (eg, sequencing device 102) described above divides the liquid in the bath 400 into two regions, a source region 402 and one or more collection regions 404. Can be used in a given configuration. Source region 402 includes fluids such as the ions, water, gels or other types of liquids described above, and several types of nucleic acid strands 406-1 and 406-2 such as DNA strands.
As shown, the collection region 404 can be formed by a collection tank 408. The collection tank 408 can isolate the liquid in the collection region 404 from the fluid in the source region 402, in which case the fluid in the source region 402 is the same or different from the fluid in the collection region 404. Good. Alternatively, the collection tank 408 is impermeable to the nucleic acid strands 406-1 and 406-2 so that the nucleic acid strands 406-1 and 406-2 are collected from the source region 402 to the collection region 404 and vice versa. It may be porous to allow fluid in the collection region 404 to flow into the source region 402 and vice versa while preventing passage through the vessel 408.
[0049]
The source region may include a number of unrelated DNA strands and a strand of a particular type of DNA (type X) having a number of known or partially known reference sequences. The array 300 can then be controlled as described above to draw the DNA strands through the openings (eg, openings 118) of the N sequencing devices 102 that operate in the array 300. Here, N is a number much larger than 1 (for example, 100 or more). As the DNA strand passes through opening 118 and is sequenced, the extraneous strand is retracted to the source region. The source region is manipulated so that this chain is removed from the vicinity of the newly opened opening 118 and the possibility of entering another opening is negligible.
[0050]
For example, type X strands with a reference sequence are counted but simultaneously rejected and returned to the source region. However, type X strands with one or several differences in the base sequence detected by the nano-openings j (j = 1 to N) are passed through the openings 118 and contain fluid collection regions Captured in a container. Any Type X strands identical to the first strand so captured are then counted and sent to the appropriate collection container. Different (or accidentally the same) non-reference sequence X strands are collected at each opening 118. When sequencing is complete, the ratio of each type of non-reference X-chain to the reference X-strand is known, and each 100% pure sample of the mutation type is collected in an individual collection tank 408. These pure samples can then be replicated by PCR and studied individually. The above methods can be used, for example, to study the rate of mutation and / or inaccurate replication in an individual's DNA and hence aging.
[0051]
All of the above devices can be modified in other ways. For example, SiO₂Si in nitrous oxide (NO) atmosphere using oxide layer in alkaline solution_ThreeN_FourCan be replaced. Further, since the DNA molecule 111 is negatively charged, the molecule 111 is placed in the opening 118 by using an electrode such as electrodes 113 and 115 to apply a positive voltage to the liquid 110 with respect to the source of the device. Can be attracted.
As mentioned above, a gel can be used in place of the liquid 110 to contain the DNA molecules. Using gel slows the movement of ions and further improves the S / N ratio. In addition, a pressure differential can be applied across the opening to control flow independent of the voltage applied between the source and drain.
[0052]
Double-stranded DNA can be similarly analyzed. Even if the double-stranded DNA is a neutral molecule, the molecule contains a charge, so that nucleotides can be distinguished by charge detection. In addition, other molecules, such as fluorescent dyes such as Hoechst dye, are single-stranded to increase / change the rigidity of the molecule, thereby facilitating insertion of the molecule into nanometer-sized openings. Can be attached to DNA. In addition, the devices described above can be used to analyze any type of individual polymer in general, so that they can be used in industries dealing with polymers such as the petroleum industry, pharmaceutical industry and synthetic fiber industry, to name a few. Can do.
[0053]
Furthermore, large size particles can be attached to a single molecule to facilitate measurement of the charge of a single molecule (eg, nucleic acid strand 111 as shown in FIG. 1). For example, gold nanoparticles can be attached to the single-stranded DNA 6. The purpose of the gold nanoparticles is to provide a solid anchor for the DNA molecule. Gold particles are easily charged and discharged. As a result, gold particles and attached DNA molecules can be manipulated by applying an electric field.
Then, the gold nanoparticles attached to the single-stranded DNA can be placed in an insulating liquid such as synthetic oil that can be used as the liquid 110 in the configuration shown in FIG. The purpose of this liquid is to allow the particles together with the DNA strands to move freely. The liquid must be insulated to avoid charge shielding by ions present in the conductive liquid. Synthetic oils are recognized as good candidates because they are highly resistant and do not form chemical bonds with single-stranded DNA.
[0054]
Next, the charge of the gold nanoparticles can be measured using a semiconductor-based charge sensor such as the device 102 shown in FIG. 1 with a floating gate or the device 226 without a gate electrode as shown in FIG. . As the charged particles approach the device, a mirror image charge is formed at the silicon / oxide interface as described above. An appropriate bias is applied to the device (eg, device 102) to operate within a threshold range that is most sensitive to any mirror charge. The induced mirror image charge then results in a higher conductivity of the device and is read out in the form of increased current. As an example, 100 DNA molecules each containing 100 bases each having one third of the electronic charge can easily be calculated to shift the threshold voltage of a 1 μm × 1 μm MOSFET having a 10 nm thick oxide by 150 mV. . This deviation can be measured by measuring the change in the drain current of the device (eg, as measured by the ammeter 114 shown in FIG. 1). The measured charge is thought to be affected by the presence or absence of charge-blocking cations and free ions in the hydrogen shell of DNA. An electric field can also be used to separate small ions from large DNA molecules.
[0055]
Further, the nanometer-sized opening types described above, such as the opening 118 of the device 102 shown in FIG. 1, can be fabricated as a well-defined square hole as shown in FIGS. 27A and 27B. To form these holes, a series of lines with appropriate width and spacing is defined in the pattern and the pattern is defined as SiO.₂Transfer to a masking material such as The same line pattern rotated 90 ° is then defined and transferred to the underlying masking material so that only the area defined by the overlapping area between the two sets of lines is removed during etching. This process provides a much better definition of the edge of the hole as compared to defining the hole in a single lithography step, as can be seen from the pattern 450 shown in FIG. The patterns shown in FIGS. 27A and 27B are manufactured by the above technique using a pattern of lines having a width of 3 μm and a spacing of 3 μm. The resulting etching mask was then used to etch pits in silicon using potassium hydroxide (KOH).
[0056]
Further reduction in line width can be achieved using electron beam lithography. For example, a 100 nm wide line pattern can be created by electron beam lithography using a Phillips 515 scanning electron microscope (SEM). Polymethyl methacrylate (PMMA) can be used as an electronic resist to achieve the pattern 460 shown in FIGS. 28A and 28B with openings 462 and can be developed with methyl isobutyl ketone / isopropyl alcohol (MIBK / IPA). . As shown, the lines are well defined and limited by the spot size of the beam used in electron beam lithography. Since a single exposure with a Gaussian beam was used, the beginning of each line is rounded. This rounding can be eliminated by using the cross-line lithography technique described with respect to FIGS. 27A and 27B. PMMA is thin SiO as shown.₂It can also be used as an etching mask to successfully transfer the pattern to the layer.
[0057]
Thus, by combining state-of-the-art electron beam lithography with the two known size reduction techniques described above, the opening 118 can be made on the (100) silicon film. A scanning transmission electron microscope (STEM) can be used to define a 10 nm line in PMMA. Cross line is SiO₂Used to make a 10 nm square hole in the mask. KOH etching can be used to etch a V-shaped pit that provides an opening of 2-4 nm on the other side of the silicon film. Thermal oxidation of silicon results in an oxide having a volume about twice that of oxidized silicon, thereby further reducing the size of the opening. This oxidation also results in the gate oxide as described above.
[0058]
Although only a few embodiments of the present invention have been described in detail above, those skilled in the art will recognize that many variations in the embodiments are possible without significantly departing from the novel teachings and advantages of the present invention. Easy to understand. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the following claims.
[Brief description of the drawings]
FIG. 1 illustrates a system for performing DNA or RNA sequencing including a DNA or RNA sequencer configured in accordance with an embodiment of the present invention.
FIG. 2 shows a plan view of the DNA or RNA sequencer shown in FIG.
FIG. 3 shows the current detector of the system shown in FIG. 1 as adenine (A), thymine (T), guanine (G) and cytosine (C) bases of the DNA or RNA sequence pass through the DNA or RNA sequencer. It is a graph which shows the example of the waveform showing the detected electric current.
4 shows a cross-sectional view of a silicon on insulator (SOI) substrate for making a DNA or RNA sequencer as shown in FIG. 1 according to an embodiment of the present invention.
5 shows a cross-sectional view of the SOI substrate shown in FIG. 4 having a shallow n-type region and a deep n-type region formed in the silicon layer and a portion of the substrate that has been cut away.
6 shows a cross-sectional view of the SOI substrate shown in FIG. 5 in which a part of the insulator is cut out and another shallow n-type region is formed in the silicon layer.
FIG. 7 is a cross-sectional view of an SOI substrate having an opening formed by etching.
FIG. 8 shows a plan view of the SOI substrate shown in FIG. 7;
9 shows a cross-sectional view of the SOI substrate shown in FIG. 9 having an oxide layer formed on the silicon layer and on the wall surface forming the opening therein.
10 shows a plan view of the SOI substrate shown in FIG. 9. FIG.
11 shows a detailed cross-sectional view of the SOI substrate shown in FIG. 7 with an oxide layer formed on the silicon layer and on the wall surface forming the opening therein.
12 shows a plan view of the SOI substrate shown in FIG. 11. FIG.
13 shows a detailed cross-sectional view of a configuration example of an opening of the SOI substrate shown in FIG.
14 shows a plan view of the opening shown in FIG. 13. FIG.
15 shows a cross-sectional view of the SOI substrate shown in FIG. 9 with holes etched in the oxide layer and metal contacts formed above the holes respectively to contact the shallow n-type region and the deep n-type region.
16 shows a cross-sectional view of the DNA or RNA sequencer shown in FIG. 1 made according to the manufacturing steps shown in FIGS.
FIG. 17 shows a plan view of a DNA or RNA sequencer having a plurality of detectors formed by a plurality of n-type regions according to another embodiment of the invention.
FIG. 18 shows a cross-sectional view of a DNA or RNA sequencer according to another embodiment of the present invention.
FIG. 19 shows a cross-sectional view of a DNA or RNA sequencer according to a further embodiment of the invention.
FIG. 20 shows a cross-sectional view of a DNA or RNA sequencer according to a further embodiment of the invention.
FIG. 21 shows a plan view of the DNA or RNA sequencer shown in FIG.
FIG. 22 is a conceptual block diagram illustrating an example of a matrix arrangement of a DNA or RNA sequencer.
FIG. 23 is a cross-sectional view of a sequencer having a metal layer instead of an intermediate semiconductor layer to realize electron tunneling.
24 shows a detailed cross-sectional view of another configuration example of the opening of the SOI substrate shown in FIG.
25 is a plan view of the opening shown in FIG. 24. FIG.
FIG. 26 illustrates a cross-sectional view of a multi-opening sequencer used with a separate liquid region.
FIGS. 27A and 27B are photographic images of opening patterns formed in a semiconductor structure. FIGS.
FIGS. 28A and 28B are photographic images of opening patterns formed in a semiconductor structure. FIGS.

Claims (31)

A system for determining the nucleic acid sequence of a nucleic acid strand, comprising :
Including at least one semiconductor device having at least one detection region having an opening through which the nucleic acid strand can pass, wherein the opening has a cross-sectional area of 100 nm or less, and the at least one nucleic acid strand is in the opening The charge of at least a component of the nucleic acid strand is configured to create a mirror image charge in the region while passing through the region, and the mirror image charge is made conductive in the region by an amount related to the charge of the component. Enough to increase
The detection region has a cross-section sufficient to allow the nucleic acid strand to enter the opening, such that the detection region will detect the charge of the component at the opening; A system comprising a region of the semiconductor device having an insulating layer defining an opening in the semiconductor device.   The system of claim 1, wherein the insulating layer comprises an oxide layer.   The system of claim 1, wherein the region of the detection region comprises an undoped semiconductor layer.   The system of claim 1, wherein the region of the detection region includes an oxide layer with the opening formed therein, the oxide layer including at least one silicon nanocrystal.   The system of claim 1, wherein the opening is non-circular. A method for manufacturing an apparatus for determining the nucleic acid sequence of a nucleic acid strand, comprising :
Providing a semiconductor structure comprising at least one semiconductor layer;
Creating a detection region having an opening through which the nucleic acid strand can pass in the semiconductor structure;
Forming an undoped semiconductor layer on a wall surface of the semiconductor layer that forms the opening to reduce the cross section of the opening, and
The opening has a cross-sectional area of 100 nm or less, and is configured such that at least a charge of a component of the nucleic acid chain creates a mirror image charge in the region while the at least one nucleic acid chain passes through the opening. And the mirror image charge is sufficient to increase the conductivity of the region by an amount related to the charge of the component.   The detection region creating step forms an oxide layer on a wall surface of the semiconductor layer that forms the opening to reduce the cross section of the opening, and the oxide layer includes at least one silicon nanocrystal. The method of claim 6, comprising the steps of:   The method of claim 6, wherein the detection region creation step further comprises forming the insulating layer comprising an oxide. A system for determining the nucleic acid sequence of a nucleic acid strand, comprising :
And including at least one structure including an insulating layer and at least one metal layer, and arranged to form at least one detection region having an opening through which the nucleic acid strand can pass, the opening having a cross-sectional area of 100 nm or less has the has the charge of at least a component of the nucleic acid strand during at least one nucleic acid strand passes through the aperture is configured to create a mirror image charge in the region, the mirror image charges, said arrangement A system, wherein an amount related to the charge of the element is sufficient to increase the conductivity of the region.   The system of claim 9, wherein the insulating layer contacts the metal layer.   The system of claim 9, wherein the structure includes two metal layers, and the insulating layer is disposed between the two metal layers. The detection region has a cross-section sufficient to allow the nucleic acid strand to enter the opening, such that the detection region will detect the charge of the component in the opening; The system of claim 9, comprising a region of the structure in which an opening is formed. 13. The system of claim 12, further comprising an excitation device adapted to generate movement in the structure to facilitate movement of the nucleic acid strand through the opening.   The system of claim 12, wherein the opening is non-circular.   The system according to claim 9, wherein the detection region includes a region of the structure in which the recess is formed in the structure such that the detection region is configured to detect a charge of the component in the recess.   The at least one structure passively detects the charge of the component without a portion of the semiconductor device that binds to or chemically reacts with the component. Item 10. The system according to Item 9.   The system of claim 9, wherein the at least one structure passively detects the single charge of the component. A method for manufacturing an apparatus for determining the nucleic acid sequence of a nucleic acid strand, comprising :
Providing a structure comprising an insulating layer and at least one metal layer;
Creating a detection region in the structure,
The detection region has an opening through which the nucleic acid strand can pass, the opening has a cross-sectional area of 100 nm or less, and at least the nucleic acid while the at least one nucleic acid strand passes through the opening. The charge of a chain component is configured to create a mirror image charge in the region, the mirror charge being sufficient to increase the conductivity of the region by an amount related to the charge of the component. Method. The component comprises a base of a nucleic acid strand;
19. The method of claim 18, wherein the creating step creates the detection region adapted to detect the charge representing the base of the nucleic acid strand. Creating an opening in the structure, the opening having a cross section sufficient to allow a portion of the nucleic acid strand to pass through, and wherein the detection region comprises the component in the opening The method of claim 18, further comprising the step of being positioned relative to the detection region so as to detect the charge representative.   21. The method of claim 20, wherein the opening is non-circular.   19. The method of claim 18, further comprising creating the recess in the structure positioned with respect to the detection area such that the detection area detects the charge representing the component in the recess. .   19. The detection region passively detects charge of the component without a portion of the semiconductor device that binds to or chemically reacts with the component. The method described.   The system of claim 18, wherein the detection region passively detects a single said charge of the component. A system for determining the nucleic acid sequence of a nucleic acid strand, comprising :
A plurality of detection devices;
A reader, and
Each of the detection devices has at least one detection region having an opening through which the nucleic acid strand can pass, the opening has a cross-sectional area of 100 nm or less, and the at least one nucleic acid strand has the opening. The charge of at least a component of the nucleic acid strand is configured to create a mirror image charge in the region while passing through the portion, and the mirror image charge is electrically conductive in the region by an amount related to the charge of the component. Enough to enhance sex,
The system wherein the reader is selectively adapted to obtain a reading from each of the detection devices, each of the readings representing each of the charges detected by the detection device. 26. At least one of the detection devices includes at least one semiconductor device having at least one of the detection regions adapted to detect a charge of a component of the nucleic acid strand proximate to the detection region. The system described in. At least one of the detection devices includes a structure including an insulating layer and at least one metal layer arranged to form at least one of the detection regions, and represents a component of the nucleic acid strand proximate to the detection region 26. The system of claim 25, adapted to detect charge.   Each of the detection devices passively detects the charge of the component without a portion of the semiconductor device that binds to or chemically reacts with the component. 25. The system according to 25.   26. The system of claim 25, wherein each of the detection devices passively detects the single charge of the component. A system for determine the nucleotide sequence of a nucleic acid strand,
Including at least one semiconductor device having at least one detection region having an opening through which the nucleic acid strand can pass, wherein the opening has a cross-sectional area of 100 nm or less, and the at least one nucleic acid strand is in the opening The charge of at least a component of the nucleic acid strand is configured to create a mirror image charge in the region while passing through the region, and the mirror image charge is made conductive in the region by an amount related to the charge of the component. The detection region is adapted to detect the charge of the component in the opening, and the region of the detection region is an oxide layer in which the opening is formed Wherein the oxide layer comprises at least one silicon nanocrystal. A method for manufacturing an apparatus for determining the nucleic acid sequence of a nucleic acid strand, comprising :
Providing a semiconductor structure comprising at least one semiconductor layer;
Creating a detection region in the semiconductor structure, wherein the detection region has an opening through which the nucleic acid chain can pass, the opening has a cross-sectional area of 100 nm or less, and the at least one nucleic acid chain is configured as the electric charge of at least a component of the nucleic acid strand to create a mirror image charge in the region while passing through the opening, the mirror image charge is the amount related to the charge of the component, Sufficient to increase the conductivity of the region;
The opening creation step forms an oxide layer on a wall surface of the semiconductor layer forming the opening to reduce the cross section of the opening, and the oxide layer includes at least one silicon nanocrystal. A method comprising the step of forming an oxide layer.

Images