Patent Application
20150316529
Not applicable.
The present invention relates to a system for analyzing the sequence of molecules using a nanochannel, and more particularly to a system and method for analyzing the sequence of molecules using a nanochannel, in which a control electrode and probe electrodes are disposed on or around a nanochannel, and the types of individual molecules in a biological polymer passing through the nanochannel are read in real time by sensing either the change in charge distribution induced by the electrical dipoles of different individual molecules or the change in current caused by the orbital energy of the individual molecules while uniformly controlling the moving speed, arrangement and direction of the individual molecules of the biological polymer passing through the nanochannel.
Reading the sequence of individual molecules of biological polymers (for example, the amino acid sequence of molecules such as polypeptides or proteins, or the sequence of DNA base molecules) is very important in the understanding of biological information processing mechanisms. As a representative example, DNA is the whole of genetic information and consists of nucleotide units. A protein is synthesized based on the sequence of nucleotides encoded in deoxyribonucleic acid (central dogma), and if DNA has a mutated nucleotide sequence different from the original nucleotide sequence, a protein cannot be synthesized or a completely different protein can be synthesized to cause serious physiological problems. Thus, examining whether DNA has a correct nucleotide sequence is very important in terms of the prevention and treatment of diseases. As a result of the human genome project, the human genetic map has been constructed, and thus pathological diagnosis and treatment at the genetic level have been increasingly activated.
There are a total of four types of nucleotides, and each nucleotide consists of an identical pentose (deoxyribose), an identical phosphate group, and one of four types of bases: adenine (A), guanine (G), cytosine (C), and thymine (T). Herein, A and G are purines having a bicyclic structure, and C and T are pyrimidines having a monocyclic structure.
A variety of DNA sequencing methods have been developed, including early methods such as the Maxam-Gilbert sequencing method and the chain-termination method, and recent methods such as the dye-terminator sequencing method. However, such methods have disadvantages in that the number of bases that are analyzed per unit time is small and in that preliminary operations such as radioactive isotope substitution or dying are time-consuming. Moreover, such methods have disadvantages in that they are costly and discharge environmental pollutants such as radioactive waste after analysis. In addition, there is a limit to the length of DNA that can be analyzed, and there is difficulty in analyzing a number of DNAs at the same time. In view of such various problems occurring in the conventional molecule sequencing methods, the recent rapid development of nanotechnology will provide potential alternative technology for real-time sequencing of molecules in combination with biotechnology. This nanobiotechnology is currently in the research and development stage, but if it is realized in the future, it will be more simple and accurate and can significantly reduce the time required for molecular sequencing, compared to the conventional chemical methods as described above.
The present invention has been made in order to solve the above-described problems of the conventional system for analysis of individual molecules of biopolymers by the use of nanotechnology, and it is an object of the present invention to provide a system for real-time analysis of the sequence of molecules, which can reduce the excessive time required for preliminary operations, can fundamentally eliminate the discharge of environmental pollutants such as radioactive waste, and can accurately analyze the sequence of individual molecules at high speed.
A system for analyzing the sequence of molecules using nanochannels according to the present invention can be used to read the sequence of individual molecules of various biopolymers, for example, polypeptides, proteins or DNA. Specifically, the system for analyzing the sequence of molecules according to the present invention comprises: at least one nanochannel having a width and height that allows the individual molecules of a biopolymer (e.g., the amino acids of protein, or the base molecules of ss-DNA) to pass therethrough without twisting or folding; at least one control electrode disposed on any one side of the nanochannel across the nanochannel and configured to align the individual molecules, which are introduced into the nanochannel, in the same direction according to the electrical or chemical properties of the individual molecules; one or more probe electrodes, one end or side of which is disposed adjacent to one side of the nanochannel in a direction perpendicular to the lengthwise direction of the nanochannel and which are configured to sense either the change in charge distribution induced by the electric dipoles of different individual molecules passing through the nanochannel or the change in current caused by a difference in the orbital energy of the individual molecules; and a measurement element configured to measure the absolute or relative value of the change in charge distribution or current sensed by the probe electrodes.
Herein, the probe electrodes may be coated with complementary molecules capable of chemically bonding with the individual molecules of the biopolymer, respectively, in order to enhance their interaction with the individual molecules of the biopolymer passing through the nanochannel. For example, when the sequence of ss-DNA base molecules passing through the nanochannel is to read, at least four probe electrodes are formed separately, and are coated with four different DNA base molecules (T, G, A, and C), respectively, so that the DNA base molecules can form a chemical bond (T-A or C-G) with complementary base molecules that are introduced into the nanochannel, thereby maximizing the sensing efficiency of the probe electrodes.
The probe electrodes may be formed of a conductive or semi-conductive material including gold, silver, copper, platinum, palladium, titanium, nickel, cobalt, graphene, graphite, or carbon nanotubes.
The control electrode may be formed of a conductive material including gold, silver, copper, platinum, palladium, titanium, nickel or cobalt, and may be disposed on or under the nanochannel or under a substrate, so that it may be applied with a specific voltage, or earthed or floated.
Alternatively, the control electrode may be formed of a material, which includes graphene, graphite or carbon nanotubes and is capable of interaction with the individual molecules (e.g., π-π energy resonance with DNA nucleotide base molecules), and it may be disposed on or under the nanochannel or under a substrate, so that it may be applied with a specific voltage, earthed or floated.
Each of the probe electrode and the control electrode is composed of a single-layer electrode or a multilayer electrode, and at least a portion of the lower portion of the single-layer electrode or the upper or lower layer of the multilayer electrodes may be coated with a dielectric layer.
Meanwhile, the measurement element may be any one of a field-effect transistor (FET), an operational amplifier, a single-electron transistor (SET), a high-frequency single-electron transistor (RF-SET), a quantum point contact (QPC) and a high-frequency quantum point (RF-QPC).
The nanochannel may be open at one side, and at least one of the probe electrode and the control electrode may be disposed on the open side of the nanochannel.
In addition, at least one of the width and height of the nanochannel may decrease continuously or stepwise from the inlet toward the downstream side thereof so as to allow the individual molecules to pass therethrough without twisting or folding.
Also, at least a portion of the inner side of the nanochannel may be coated with a dielectric layer.
In an embodiment of the present invention, the measurement element may be electrically connected with the probe electrode through an extended gate, and may be at an atmosphere temperature lower than the atmosphere temperature of the nanochannel.
Further, the measurement element may be formed integrally on a substrate having the nanochannel formed thereon.
The system of the present invention may comprise one or more probe electrode pairs, each consisting of two opposite probe electrodes that are located at the two opposite sides of the nanochannels, respectively, and the probe electrode pairs may be connected to different measurement elements.
A method for analyzing the sequence of molecules using a nanochannel according to the present invention comprises the steps of: moving a biopolymer in a nanochannel by electrophoresis or a difference in pressure of a fluid; applying a voltage to a control electrode formed on or under the nanochannel or under a substrate having the nanochannel formed thereon, or connecting the control electrode to an earth, or floating the control electrode, to control the direction of individual molecules of the biopolymer (e.g., nucleotide bases included in ss-DNA); inducing a change in charge distribution of a probe electrode by the individual molecules; and transferring the change in charge distribution to a measurement element to read the type of individual molecules.
Alternatively, a method for analyzing the sequence of molecules using a nanochannel according to the present invention comprises the steps of moving a biopolymer in a nanochannel by electrophoresis or a difference in pressure of a fluid; applying a voltage to a control electrode formed on or under the nanochannel or under a substrate having the nanochannel formed thereon, or connecting the control electrode to an earth, or floating the control electrode, to control the direction of individual molecules of the biopolymer (e.g., nucleotide bases included in ss-DNA); tunneling the energy levels of the individual molecules through a probe electrode pair consisting of two opposite probe electrodes; and sensing a change in the tunneling currents by a measurement element connected to the probe electrode pair to read the type of individual molecules.
Alternatively, a method for analyzing the sequence of molecules using a nanochannel according to the present invention comprises the steps of moving a biopolymer in a nanochannel by electrophoresis or a difference in pressure of a fluid; applying a voltage to a control electrode formed on or under the nanochannel or under a substrate having the nanochannel formed thereon, or connecting the control electrode to an earth, or floating the control electrode, to control the direction of individual molecules of the biopolymer (e.g., nucleotide bases included in ss-DNA); interacting the individual molecules with a single-layer probe electrode or the lower-layer electrode of a multilayer probe electrode, formed on the nanochannel; and sensing either a change in current of the single-layer electrode or a change in current of the lower-layer electrode of the multilayer probe electrode, caused by a change in a voltage applied to the upper layer electrode, by a measurement element connected to the single-layer electrode or the lower layer electrode of the multilayer probe electrode, to read the types of individual molecules.
In a method that is applied to all the methods for analyzing the sequence of molecules using the nanochannel, a plurality of probe electrodes or probe electrode pairs having the same configuration may be formed per nanochannel, so that the sequence of individual molecules of a biopolymer (e.g., ss-DNA or polypeptide) that passed through the nanochannel can be independently read a plurality of times, thereby increasing the reliability of analysis while greatly reducing the time required for analysis. This is the most important key element in the implementation of the present invention, and it is to be understood that, as the number of the plurality of probe electrodes increases, the speed and reliability of analysis of the base sequence increase. However, all the probe electrodes should be disposed within the range of the nanochannel.
In a method that is applied to all the methods for analyzing the sequence of molecules using the nanochannel, the probe electrodes may be coated with complementary molecules capable of chemically bonding with the individual molecules, respectively, which pass through the nanochannel, in order to enhance their interaction with the individual molecules. For example, if the sequence of ss-DNA base molecules passing through the nanochannel is to read, at least four independent probe electrodes may be formed, and may be coated with four different base molecules (T, G, A, and C), respectively, so that they can form a chemical bond (T-A or C-G) with complementary base molecules moving along the channel, thereby maximizing the sensing efficiency.
According to the present invention, a control electrode is disposed on or under a nanochannel, so that the moving speed, arrangement and direction of the individual molecules of a biopolymer passing through the nanochannel are maintained uniformly by the control electrode. In addition, the change in charge distribution or current induced by the molecules passing through the nanochannel is sensed by one or more probe electrodes, and the type of each of molecules is analyzed in real time by a measurement element. Thus, the present invention has an advantage in that the sequence of individual molecules can be precisely analyzed at high speed without causing environmental pollution. Particularly, if ss-DNA base molecules are to read, at least four independent probe electrodes may be formed, and the probe electrodes may be coated with four different types of DNA base molecules, respectively, so that the probe electrodes can interact with complementary base molecules passing through the nanochannel, thereby maximizing the sensing efficiency and reliability.
10: base sequence analysis system; 20: ss-DNA;
50: substrate; 100: nanochannel;
200: probe electrode; 200A: probe electrode coated with A;
200G: probe electrode coated with G;
200C: probe electrode coated with C;
200T: probe electrode coated with T;
210: single-layer electrode;
220: multilayer electrode; 222: lower layer electrode;
224: insulating layer; 225: upper layer electrode;
300: control electrode; 400: measurement element;
410: quantum dot; 411: source;
412: drain; 413: first gate;
414: second gate; 420: extended gate.
While this invention may be embodied in many different forms, there are described in detail herein a specific preferred embodiment of the invention. This description is an exemplification of the principles of the invention and is not intended to limit the invention to the particular embodiment illustrated.
Hereinafter, preferred embodiments of a system 10 for analyzing the sequence of individual molecules according to the present invention will be described in detail with reference to the accompanying drawings.
In the following description of embodiments of the present invention, the detailed description of known elements obvious to those skilled in the art will be omitted so as not to obscure the gist of the present invention.
In the drawings, the thickness of lines or the size of constituent elements may be exaggerated for the clear understanding and convenience of description. Terms such as before, after, upper, lower, left, right, inner and outer, which indicate relative locations, are based on the direction shown in the drawings.
The system for analyzing the sequence of molecules using a nanochannel according to the present invention can be used to read the sequence of individual molecules of various biopolymers such as polypeptides, proteins or DNA (for example, the sequence of amino acid molecules of protein, the sequence of base molecules of DNA). In a specific embodiment, the system of the present invention may be used to analyze the sequence of base molecules of DNA as described below.
As shown in
The fundamental function of the inventive system having the above-described configuration is as follows. Either the change in charge distribution induced by the electric dipoles of different nucleotides of a single-stranded DNA (ss-DNA) 20 passing through the nanochannel or the change in current caused by a difference in orbital energy of the nucleotides is sensed by one or more probe electrodes 200, thereby analyzing the base sequence of the DNA. Separately from the probe electrode 200, the control electrode 300 is disposed on the nanochannel 100 in order to fix or align the bases of nucleotides in a uniform direction and control the moving speed of the bases, thereby increasing the accuracy and efficiency of analysis of the base sequence.
Hereinafter, each element of the system of the present invention will be described in detail. The nanochannel 100 has a width and height that allows the ss-DNA 20 to pass therethrough without twisting or folding. Generally, the width and height of the nanochannel 100 range from 0.1 nm to several hundred nm, and the ss-DNA 20 passes through the nanochannel 100 by electrophoresis or the difference in pressure of a fluid.
When the ss-DNA is used, the bases of the DNA are exposed so that either the change in potential induced by the electric dipoles of different nucleotides or the change in electric current caused by a difference in the orbital energy of the nucleotides can be sensed. Because one strand of double-stranded DNA (ds-DNA) has a sequence complementary to that of another strand, it is possible to analyze the base sequence of the ss-DNA 20.
The nanochannel 100 has a width and height that allows the ss-DNA 20 to pass therethrough without twisting or folding. As can be seen in
Meanwhile, the control electrode 300 functions to align nucleotides in the same direction and control the moving speed of nucleotides during passage through the nanochannel. The control electrode 300 can be disposed on or under the nanochannel 100 or under a substrate 50 having the nanochannel 100 formed thereon in such a manner that it goes across the nanochannel 100. For example,
Herein, the use of the electrical properties of the nucleotides is the use of the negatively charged properties of phosphate groups present in the backbone of the ss-DNA 20. The phosphate groups of nucleotides have a negative charge. Thus, when a negative voltage is applied to the control electrode 300 disposed in the same plane as the probe electrode 200 or when the control electrode 300 is earthed, the phosphate groups will receive a repulsive force by the negative charge of the control electrode 300, and thus bases located opposite the phosphate groups (with respect to pentose located at the center) will be aligned to face the probe electrode 200. On the contrary, even when the control electrode 300 is disposed opposite the probe electrode 200 and a positive voltage is applied thereto, the same effect can be obtained. In addition, it is possible to float the control electrode 300.
The control electrode 300 as described above may be made of a conductive material including gold, silver, copper, platinum, palladium, titanium, nickel or cobalt, and may be composed of a single-layer electrode or a multilayer electrode, like the probe electrode 200.
Moreover, the use of the chemical properties of nucleotides to align is the use of the interaction (for example, p-p orbital interaction) between the bases of the nucleotides and graphene (or graphite or carbon nanotubes) that is the material of the control electrode. In other words, when the control electrode 300 is formed of a material, such as graphene, graphite or carbon nanotubes, which can interact with the bases of nucleotides, the direction of bases of nucleotides that pass below or above the control electrode 300 will be maintained uniformly by their interaction with the control electrode 300.
Furthermore, at least one probe electrode 200 is disposed adjacent to any one surface of the nanochannel 100 in a direction perpendicular to the lengthwise direction of the nanochannel 100. The probe electrode 200 functions to sense either the change in charge distribution induced by the dipole moment of different nucleotides of the ss-DNA 20 passing through the nanochannel 100, or the change in current caused by a difference in the orbital energy of the nucleotides. In other words, the probe electrode 200 refers to an electrode capable of sensing individual nucleotides.
Different nucleotides have different electric dipoles attributable to the respective charge distributions, and the change in charge distribution caused by the electric dipoles can be sensed by the probe electrode 200, thereby reading the type of nucleotide. As shown in
The probe electrode 200 may be formed of a conductive or semi-conductive material including gold, silver, copper, platinum, palladium, titanium, nickel, cobalt, graphene, graphite, or and carbon nanotubes, which can transmit electrical signals.
In alternatives to the method of sensing the change in charge distribution induced by the electric dipole, it is also possible to analyze the base sequence of the ss-DNA 20 using the change in electric current caused by the orbital energy characteristics of nucleotides.
In a first alternative analysis method, a tunneling current in a direction perpendicular to the direction in which nucleotides pass through the nanochannel is measured using a probe electrode pair consisting of two opposite probe electrodes located at the two opposite sides of the nanochannel, respectively (
In another analysis method, the probe electrode 200 (
However, the single-layer electrode 210 or the lower layer electrode 222 of the multilayer electrode 220 should be formed of a material capable of interacting with the orbital energy of nucleotide bases. For example, it should be formed of a material such as graphene, graphite, or carbon nanotubes. Between the lower layer electrode 222 and the upper layer electrode 225, an insulating layer 224 that insulates these electrodes from each other is formed. The probe electrode 200 may be composed of the single-layer electrode 210 or the multilayer electrode 220, and at least a portion of the top of the single-layer electrode 210 or the upper and lower layers of the multilayer layer electrode 220 may be coated with a thin dielectric layer. The dielectric layer is formed for the purposes of providing electrical insulation and increasing the sensitivity of measurement.
For the same purposes, at least a portion of the inner surface of the nanochannel 100 may also be coated with a dielectric layer. Particularly, it is effective to form a dielectric layer at the boundary between the probe electrode 200 and the nanochannel 100.
It is to be understood that the above-described configuration of the single-layer electrode, the multilayer electrode or the dielectric layer may, if necessary, be modified in various ways.
Meanwhile, as shown in
With respect to the measurement element 400, either the change in potential induced by the electric dipoles of nucleotides, or the change in current caused by a difference in orbital energy, is sensed by the probe electrode, and the absolute value or relative value of this change is measured by the measurement element 400 electrically connected to the probe electrode 200. In other words, the measurement element 400 can measure the changes in charge distribution and current, which change depending on the type of nucleotide, thereby determining the type of nucleotide.
The measurement element 400 that is used in the present invention may be composed of a field effect transistor (FET), an operational amplifier, a single electron transistor (SET) or a quantum point contact (QPC).
Also, in order to further increase the measurement speed and sensitivity of the measurement element 400, a high-frequency (RF) resonance circuit may be attached to any one or both of the source 411 or drain 412 of the measurement element 400, so that high frequency can be applied to thereby measure a change in high-frequency transmission or reflection. In addition, an additional amplifier may be disposed at a position closest to the source 411 or drain 412 of the measurement element 400, so that the signal of the additional amplifier can be sensed. The measurement element 400 that employs high frequency may be, for example, a high-frequency single-transistor transistor (RF-SET) or a high-frequency quantum dot contact (RF-QPC).
In addition, the measurement element 400 may be configured such that it is electrically connected to the probe electrode 200 through an extended gate 420 and is at an atmosphere temperature lower than the temperature surrounding the nanochannel 100. This embodiment is illustrated in
In addition, the sequence analysis system 10 according to the present invention may be configured to have a simplified structure by forming the nanochannel 100 on the substrate 50 and forming the measurement element 400 on the substrate 50 (see
Meanwhile, each of the nanochannel 100, the probe electrode 200, the control electrode 300 and the measurement element 400, which are included in the sequencing system 10 of the present invention, may be one or more in number. For example,
The structures having the plurality of probe electrodes (
In addition, in a method that is applied to the methods for analyzing the sequence of molecules using a nanochannel, the probe electrodes may be coated with complementary molecules capable of chemically bonding to ss-DNA base molecules passing through the nanochannel, in order to enhance their interactions with the ss-DNA base molecules. This method can be applied to the case in which either the change in charge distribution induced by the electric dipoles of different nucleotides of a ss-DNA or the change in current caused by a difference in the orbital energy of nucleotides is insignificant so that noise cannot be overcome by the probe electrode. For example, as shown in
Among the probe electrodes 200 shown in
In another method that is applied to the method of analyzing the sequence of base molecules using a nanochannel and a plurality of probe electrode pairs, a specific voltage is applied through the pairs of opposite probe electrodes so as to allow resonant tunneling between the probe electrode pair and any one base molecule among the four different base molecules of the ss-DNA passing through the nanochannel. For example, as shown in
Among the four probe electrode pairs 200 shown in
Although the preferred embodiments of the present invention have been disclosed for illustrative purposes with reference to the accompanying drawings, those skilled in the art will appreciate that various modifications, additions and substitutions are possible. For example, a plurality of nanochannels may be formed in parallel on a single substrate, and various parts of a single ss-DNA may be passed through the nanochannels and analyzed individually at the same time using the base sequence analysis system of the present invention. In this case, the time required to read the sequence of all the individual molecules of the ss-DNA.
This completes the description of the preferred and alternate embodiments of the invention. Those skilled in the art may recognize other equivalents to the specific embodiment described herein which equivalents are intended to be encompassed by the claims attached hereto.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2012-0035945 | Apr 2012 | KR | national |
This is a national stage application of PCT/KR2012/003154, filed on Apr. 25, 2012, claiming priority to KR10-2012-0035945, filed on Apr. 6, 2012
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR12/03154 | 4/25/2012 | WO | 00 | 9/22/2014 |
