KR20130118573A - Method and analysis system for real-time molecular sequencing with stacked nanoribbon - Google Patents

Abstract

PURPOSE: A real-time molecular sequence analysis system and analysis method using laminate nano ribbon can maximize current changes in a nano ribbon to maximize a sensing efficiency and reliability, and can analyze a base sequence comprised of 4 base molecules through one-time movement of ss-DNA. CONSTITUTION: A real-time molecular sequence analysis system using the laminate nano ribbon (200) comprises the measuring electrode (300), the insulating layer (400), the substrate (500) and the nano pore (100). Said nano ribbon has a pre-determined width in which current variation is induced by mutual interactions with the most adjacent unit molecule among unit molecules forming the biopolymer. Said measuring electrodes are formed respectively at both ends in a length direction of said nano ribbon. Said insulating layer is formed on an upper and a lower faces of said nano ribbon to electrically insulate said nano ribbon. Said substrate mechanically supports the nano ribbon in which said insulating layer and measuring electrodes are formed. Said nano pore vertically passes through said substrate, and a center of the insulating layer and the nano ribbon.

Description

Method and Analysis System for Real-Time Molecular Sequencing with Stacked Nanoribbon}

The present invention relates to a real-time molecular sequence analysis system using stacked nanoribbons, and more particularly, at least four nanoribbons are stacked on a substrate with an insulating layer interposed therebetween, the substrate, the insulating layer and the nano A nanopore is formed penetrating the center of the ribbon in a vertical direction, and the nanoribbons are formed by the interaction between the unit molecules and the nanoribbons constituting the biopolymer while the biopolymer passes through the nanopore. A molecular sequence analysis system that detects the change of the unit spherical component in real-time by sensing the current flowing through the measured and measured change through a measurement electrode formed at each end of the longitudinal length of the nanoribbon, and It's about how.

Decoding the unit molecule sequence (amino acid molecule sequence of the protein, DNA base molecule sequence) constituting the biological polymer means decoding the biometric information, the molecular genetic understanding can make early diagnosis of human disease It is very important in that it is. For example, DNA is composed of nucleotide units, and each nucleotide has the same one deoxyribose and phosphate group and has four different bases: adenine (A), guanine (G), and cytosine (C). ), Thymine (T) is present, and A and G are Purine series having two cyclic structures, and C and T are Pyrimidine series having one cyclic structure. Based on the nucleotides recorded in the deoxyribonucleic acid, messenger RNA (mRNA) is transcribed and proteins are synthesized through the binding of amino-acids in ribosomes. If there is a mutated nucleotide sequence different from the original nucleotide sequence, protein synthesis may not be possible or abnormal proteins may be synthesized and expressed, resulting in physiological problems. For this reason, the meaning of deciphering the information possessed by DNA is very important from a pathological point of view for the early diagnosis and treatment of diseases.

Various methods have been developed for DNA sequencing, ranging from early Maxam-Gilbert sequencing to Dye-Terminator sequencing. However, these methods limit the length of DNA that can be analyzed per unit time, and require a long preparation process such as repeating DNA amplification, radioisotope substitution and staining for accurate analysis. In addition, the analysis of the entire DNA through the pieces of DNA is done through the processing of a lot of data, and requires a lot of money because of the time required and the chemicals required for analysis. The problem faced by this analytical method is that various solutions have been proposed through the convergence of rapidly developed nanotechnology and biotechnology, and can provide infinite potential technology. As a representative example, electrical analysis methods have been proposed by applying a solid element that can be used semi-permanently by breaking away from conventional chemical reaction methods. Successful implementation of these technologies can reduce the time required for preparation and analysis, reduce waste by reducing chemicals, and enable simpler and more accurate analysis.

The present invention is to solve the problems of the existing unit molecular sequence analysis system using nanotechnology, to reduce the excessive time and harmful chemicals that are invested in the preliminary work for molecular sequence analysis, and compared to the wasteful conventional method Unlike other semi-permanent solid-state devices, it is possible to analyze real-time unit molecules at high speed, and to provide a cheaper molecular sequence analysis system.

Real-time molecular sequence analysis system using laminated nanoribbons according to the present invention can be used to decode a variety of biopolymers. For example, it is possible to decode the unit molecule constituting the protein and DNA, thereby understanding the biometric information. Specifically, the change in charge distribution is induced by the electric dipoles of unit molecules forming adjacent biopolymers, and thus the nanoribbons of a predetermined width and the top surface of the nanoribbons, which induce a change in edge current flowing through the edges, are induced. An insulating layer formed on an underside to insulate the nanoribbons from the surrounding electrolyte and other nanoribbons; Measuring electrodes formed at both ends of the nanoribbons at both ends of the nanoribbons and electrically connected to each other, applying a predetermined voltage, and measuring a change in current by combining with a current measuring device; And a substrate that mechanically supports the nanoribbon on which the insulating layer and the measurement electrode are formed; And nanopores that can pass through the biopolymer without twisting or overlapping and have a diameter and penetrate vertically through the center of the substrate, the insulating layer, and the nanoribbon; . Wherein the complementary molecules (complementary) that can induce complementary bonds to maximize the current change by increasing the interaction with the unit molecules of the biopolymer through the nanopore to each nanoribbon of the formed nanopore molecules can be coated. For example, at least four nanoribbons are stacked on a substrate with an insulating layer interposed to detect all four types of nucleotides that are structural units during single-stranded DNA (ss-DNA) movements. Each nanoribbon inside the nanopore formed through the center perpendicularly to the center thereof and coated with one of the different bases A, G, T, and C, and complementary to the DNA base moving into the nanopore (AT or GC). By maximizing the charge distribution change of the nanoribbon, it is possible to maximize the detection efficiency and reliability of the current change flowing through the edge.

In addition, by combining a hydrogen atom or a nitrogen atom to a carbon atom having a dangling bond of the nanopore and the nanoribbon edge, the interaction between the structural unit molecules of the biopolymer and the nanoribbon can be maximized.

The structure for decoding the unit molecule sequence of the biopolymer may include a junction structure of a zig-zag graphene nanoribbon and an insulation layer or a two-dimensional topological insulators nanoribbon and an insulation layer. It may be a junction structure, the diameter of the nanopores penetrating the junction structure width and the center in the vertical direction each have a nanometer to several tens of nanometers. Here, two-dimensional topological insulators are characterized in that the nanoribbons have an almost insulating interior while their edges are conductive.

The measuring electrode is made of a conductor including gold, silver, copper, platinum, palladium, titanium, nickel, and cobalt, and is electrically connected at both ends in the longitudinal direction of the nanoribbon, and applies a predetermined voltage to the nanoribbon. A predetermined current flows and the current change due to the interaction between the unit molecules and the nanoribbons can be measured.

The insulating layer may be formed of an electrical insulator including a silicon oxide film, a silicon nitride film, an aluminum oxide film, a silver oxide film, a zinc oxide film, and a hafnium oxide film, and may insulate the nanoribbon from the surrounding electrolyte and other nanoribbons.

On the other hand, the molecular sequence analysis method using the laminated nanoribbon according to the present invention, by applying a predetermined voltage to the measurement electrode formed on each nanoribbon to flow a predetermined current through the nanoribbon; And applying a voltage or a fluid pressure between one side and the other side of the substrate to allow the biopolymer to pass through the nanopores; And the unit molecules forming the biopolymer interact with the nanoribbons to change a current flowing through the nanoribbons; Sensing current flowing through the nanoribbon; And combining-analyzing current data flowing through the nanoribbons to determine the sequence of unit molecules. .

In addition, by adding a stack of the next nanoribbons, one nanopolymer may be moved once, and analyzed several times through each nanoribbon, thereby greatly reducing the time required while increasing reliability.

According to the present invention, a nanoribbon is stacked on a substrate with an insulating layer interposed therebetween and moves nanopores vertically through the center of the substrate, the insulating layer and the nanoribbon by voltage or fluid pressure pressure on one side and the other side of the substrate. Solids escaped from conventional chemical methods by analyzing the current change of nanoribbons from the unique electric dipoles of biopolymers in real time through measuring electrodes electrically connected to nanoribbons and analyzing the identity of each unit molecule. The device can significantly reduce the time required and the use of chemicals, and has the advantage of being able to analyze unit molecule sequences with high speed and precision. In particular, in order to analyze the sequence of ss-DNA, at least four layers of nanoribbons are sandwiched between insulating layers, and four different DNA base molecules (A and G) are present in each nanoribbon of nanopores formed at the center thereof. , T, C) can be maximized the detection efficiency and reliability by maximizing the current change of the nanoribbon through complementary bonding with base molecules moving into the nanopore, and through a single ss-DNA movement 4 All nucleotide sequences consisting of two base molecules can be analyzed.

1 is a view showing the overall configuration of a single nucleotide sequence analysis system applied to the DNA base molecule sequence translation as an embodiment of the invention.
2 is a cross-sectional view taken along the line AA of FIG. 1.
FIG. 3 shows measured data predictable in real time using nanoribbons coated with four different bases applicable to the present invention. FIG.

Hereinafter, with reference to the accompanying drawings will be described in detail a preferred embodiment of a unit molecule analysis system according to the present invention.

In describing the embodiments of the present invention, a description of well-known structures that can be understood by those skilled in the art will be omitted so as not to obscure the gist of the present invention.

In addition, when referring to the drawings, it should be considered that the thickness of the lines shown in the drawings, the size of the components, etc. may be exaggerated for the sake of clarity and convenience of the description. The term is based on the direction shown in the drawings unless otherwise noted.

The real-time molecular sequence analysis system using the stacked nanoribbons according to the present invention decodes the unit molecule sequence (for example, amino acid molecules of proteins, or base molecule sequences of DNA, etc.) constituting various biopolymers such as proteins or DNA. It can be used to As a specific example, specific contents applied to DNA nucleotide sequence analysis will be described below.

1 and 2, the real-time molecular sequence analysis system using the stacked nanoribbons according to the present invention is largely nanopore 100, nanoribbons (200: 200A, 200G, 200T, 200C), measuring electrode ( 300, an insulating layer 400, and a substrate 500.

Referring to the basic function of the present invention having the above configuration, a predetermined voltage is applied to the measurement electrode 300 to flow a predetermined current through the nanoribbons 200, and the other side of the substrate 500 Around the nanopores 100 from different nucleotide-specific electric dipoles that make up the single-stranded DNA (ss-DNA) 10 that pass through the nanopores 100 by a voltage or fluid pressure applied therebetween. By inducing charge distribution of the nanoribbon and analyzing the change of the edge current flowing through the edge of the nanoribbon 200, the identity of the nucleotides ss-DNA 10 can be analyzed. In this case, each of the nanoribbons inside the nanopore 100 is coated with any one of different bases A, G, T, and C, and is complementary to the nucleotides forming the ss-DNA 10 passing through the nanopore 100 ( AT or GC) to maximize the interaction with the nanoribbon 200 to maximize the current change through the edge of the nanoribbon 200.

The constitution of the present invention as described above will be described in detail for each constituent element in turn as follows.

First, the nanopores 100 are formed by vertically penetrating the centers of the substrate 500, the insulating layer 400, and the nanoribbons 200, and pass through the ss-DNA 10 without twisting or overlapping. It has a diameter that can be in the range of usually less than 2nm. The ss-DNA 10 passes through the nanopores 100 by a voltage or a fluid pressure applied between one side and the other side of the substrate 500.

Here, ss-DNA (10) is used to induce a change in the charge distribution of the nanoribbons 200 from the electric dipoles of different nucleotides by exposing the base to the outside, DNA is a double helix structure for one strand Since the other strand has a complementary sequence, it is possible to analyze the nucleotide sequence with one ss-DNA (10).

The nanoribbons 200 are used to detect the actual nucleotides, and the distance between the different bases forming the ss-DNA 10 is 5 Å or less, and the range of the thickness of the nanoribbons 200 must belong to the nucleotides. When analyzing, it is possible that there is no great interference with other adjacent nucleotides. In addition, in order to induce a change in the effective current of the nanoribbons 200 from the base electric dipole should have a width of less than 10nm. In addition, a zig-zag graphene or two-dimensional topological insulator satisfying the above conditions may serve as the nanoribbons 200. In the nanoribbons shown in FIGS. 1 and 2, 200A is coated with deoxyribonucleotide (dATP) having adenine or adenine as a base on the nanopore 100, and 200G is similarly deoxyribonucleotide having a guanine or guanine as a base. dGTP) is coated with thymine or deoxyribonucleotide (dTTP) with thymine as base, and 200C is coated with deoxyribonucleotide (dCTP) with cytosine or cytosine as base. Here, when the ss-DNA 10 passes through the nanopores 100, the nanoribbons through the complementary bond (AT or CG) of the base constituting the ss-DNA 10 and the base coated on the nanoribbons 200. The change of the charge distribution is maximized, and thus the change of the edge current of the nanoribbon is maximized. In addition, by combining hydrogen atoms or nitrogen atoms with carbon atoms having dangling bonds at the edges of the nanopores 100 and nanoribbons 200 in addition to the coating of the nucleotides, You can also maximize interaction.

The measuring electrode 300 may be formed of a conductor including gold, silver, copper, platinum, palladium, titanium, nickel, and cobalt, and is electrically connected to both ends of the nanoribbons 200 in the longitudinal direction.

In addition, the stacked nanoribbons 200 are formed on the upper and lower surfaces of the nanoribbons 200 for the analysis of each nucleotide, and an insulating layer 400, which is an electrical non-conductor, is formed in a length direction with one nanoribbon 200. The measuring electrodes 300 formed at both ends are electrically insulated from an external electrolyte or other nanoribbons 200.

The nanoribbons 200 on which the insulating layer 400 and the measuring electrode 300 are formed are mechanically supported by the substrate 500.

FIG. 3 shows in real time measurement data predictable through the edge current modulation of the four independent nanoribbons coated with ss-DNA 10 passing through nanopore 100 with respective different bases. These four data can be combined-analyzed in real time to read the sequence AGTCAGTC of the ss-DNA 10 that has passed through the nanochannel.

10: ss-DNA
100: nanopores
200: nanoribbon
200A: A-coated nanoribbons
200G: G-coated nanoribbons
200T: T-coated nanoribbons
200C: C coated nanoribbons
300: measuring electrode
400: insulation layer
500: substrate

Claims (10)

A nanoribbon having a predetermined width at which a change in current is induced by interaction with the nearest unit molecule of the biomolecules;
Measuring electrodes formed at both ends of the nanoribbon in the longitudinal direction;
An insulating layer formed on the top and bottom surfaces of the nanoribbons to electrically insulate the nanoribbons;
A substrate that mechanically supports the nanoribbon on which the insulating layer and the measurement electrode are formed;
Nanopores penetrating the center of the substrate, the insulating layer and the nanoribbons in a vertical direction;
Real-time molecular sequence analysis system using laminated nanoribbons consisting of. The method of claim 1,
At least four nanoribbons are stacked on the substrate with an insulating layer interposed therebetween, wherein the stacked nanoribbons are composed of nanopores penetrating the center of the substrate, the insulating layer and each of the nanoribbons in a vertical direction. Real-time molecular sequence analysis system using. 3. The method of claim 2,
By coating the nanoribbons with different unit molecules of biopolymers, inducing complementary bonding with unit molecules passing through the nanopores, real-time molecules using stacked nanoribbons, maximizing the current change of the nanoribbons Sequence Analysis System. The method of claim 1,
The nanoribbons include zig-zag graphene or two-dimensional topological insulators, and the inside of the nanoribbon is almost insulating and the edges are conductive. Real-time molecular sequence analysis system using laminated nanoribbons. The method of claim 1,
The measuring electrode is made of a conductor comprising gold, silver, copper, platinum, palladium, titanium, nickel, cobalt, and is electrically connected to the nanoribbon, real-time molecular sequence analysis system using a laminated nanoribbon . The method of claim 1,
The insulating layer is formed of an electrical insulator including a silicon oxide film, a silicon nitride film, an aluminum oxide film, a silver oxide film, a zinc oxide film, and a hafnium oxide film, and insulates the nanoribbons from the surrounding electrolyte and other nanoribbons. Real-time molecular sequence analysis system using stacked nanoribbons. The method of claim 1, wherein
Real-time molecular sequence analysis system using a laminated nanoribbon, characterized in that for bonding a hydrogen atom or a nitrogen atom to a carbon atom having a dangling bond formed on the edge of the nanopore and nanoribbon. The method of claim 1,
The width of the nanoribbon and the diameter of the nanopore is a real-time molecular sequence analysis system using a stacked nanoribbon, characterized in that each of 1 nanometer to several tens of nanometers. Applying a predetermined voltage to the measurement electrodes formed on each nanoribbon such that a predetermined current flows through the nanoribbon;
Applying a voltage or fluid pressure between one side and the other side of the substrate to allow the biopolymer to pass through the nanopores;
Changing the current flowing through the nanoribbons by interacting with the nanoribbons unit molecules forming the biopolymer;
Sensing a current flowing through the nanoribbon; And
Combining and analyzing current data flowing through each nanoribbon to determine the sequence of unit molecules;
Real-time molecular sequence analysis method using a laminated nanoribbon consisting of. The method of claim 9,
Real-time molecular sequence analysis method using the stacked nanoribbons, characterized in that the current flowing through the nanoribbon is almost the edge current (edge current).

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