KR101297226B1 - Device and method for ultrafast dna sequencing based on graphene nanoribbons - Google Patents

Abstract

The present invention relates to a device and a method for analyzing a base sequence such as DNA, and more particularly, the aromatic interaction and type of base of graphene nanoribbon and DNA base when DNA passes under the graphene nanoribbon Based on the specificity of the electrical conductivity according to the present invention relates to a device and method that can quickly and accurately analyze the nucleotide sequence of DNA.
A sequencing device using the graphene nanoribbons of the present invention includes a substrate on which a nano groove is formed, a graphene nanoribbon placed on the substrate, a current application electrode connected to both ends of the graphene nanoribbon, and the substrate. And a gate voltage application electrode connected below. The method of analyzing the base sequence using the analysis device includes applying a bias voltage between both electrodes of the graphene nanoribbons and applying an alternating gate voltage to the gate electrode, and passing through the graphene nanoribbons through the nano grooves. Towing the DNA to be analyzed so as to detect the current flowing through the graphene nanoribbon in real time, and analyzing the relationship between the current and the gate voltage of the graphene nanoribbon over time. As a result, genetic information can be decoded as quickly as possible within hours.

Description

Ultrafast sequence analysis device and method using graphene nanoribbons {DEVICE AND METHOD FOR ULTRAFAST DNA SEQUENCING BASED ON GRAPHENE NANORIBBONS}

The present invention relates to a device and a method for analyzing a base sequence such as DNA, and more particularly, the aromatic interaction and type of base of graphene nanoribbon and DNA base when DNA passes under the graphene nanoribbon Based on the specificity of the electrical conductivity according to the present invention relates to a device and method that can quickly and accurately analyze the nucleotide sequence of DNA.

Genetic information means all the information necessary for the maintenance of life and self-replication of a living organism, which is determined by the structure of DNA which is the body of the gene. DNA consists of four bases: adenine (A), guanine (G), cytosine (C), and thymine (T). The sequence information contains genetic information. Therefore, the interpretation of genetic information is the task of analyzing the base sequence in DNA.

Human genes consist of about three billion pairs of bases. To decode this, an initial investment of about 13 years and about $ 3 billion was invested, completing the human genome map in 2001. With the successful completion of the Human Genome Project in 2003, a faster and easier way to decipher genetic information is now required. Such fast and inexpensive genetic information decryption method is the most essential in the future bio industry such as personalized medicine (personalized medicine). The National Institutes of Health (NIH) announced in 2004 that it aims to analyze the human genome with time of day and $ 1,000.

Early first-generation methods were based on Sanger's method. Sanger's method basically breaks down genes into small units, chemically amplifies them, then chemically labels them, and then analyzes each of the broken genetic units through electrophoresis. That's how. At this time, the process of cutting-amplifying-labeling requires enormous time and cost, and the electrophoresis method also requires considerable time. Therefore, a new method is needed that does not require this work.

The cyclic-array method, which is currently used, takes several weeks to analyze the human genome, and is a second generation method that slightly improves on the previous Sanger method. Attempts to massively parallelize using microarrays have significantly reduced the analysis time compared to Sanger's method. However, this method also requires a cut-amplify-labeling method and still requires considerable cost and time in the process.

With the rapid development of nanotechnology, sequencing using nanodevices has emerged. A typical method is to use a nanopore to analyze the current along the base passing through it. However, this method fails to control the structural movement of the base due to the non-uniformity of the nanopores, resulting in severe fluctuations in the measured current. This makes it impossible to analyze each base even if the current is measured. Therefore, there is a need for a technique to reduce the fluctuation of the base.

In 2005, Di Ventra's research group in the United States showed the possibility of distinguishing each base by modeling the base passing through the nanopores and theoretically calculating the current through the base. The range of currents due to the structural change (rotation) of the base as it passes through the nanopores is calculated to further increase the feasibility of the method through the nanopores. However, in 2006, a simulation of the actual DNA flowing through the nano-pores was performed using a computer simulation method, and the distribution of the current flowing through the base was analyzed, and a considerable overlap was found. This is due to the structural movement of the base's nanopores. Therefore, this structural movement must be limited for the nanohole method to become a reality. In addition, since the experiment is performed at the nano level, accurate analysis can be performed only if the signal-to-noise ratio (SNR) can be increased.

The present invention is to design a sequencing device using a graphene nanoribbon, which can be much more accurate than the conventional technology using the nano-pores.

The present invention also provides a breakthrough sequencing device using graphene nanoribbons, and provides a new method for analyzing the measurement results of the device.

In the context of the present invention, the inventors' paper SK Min et al., “Fast DNA sequencing with a graphene-based nanochannel device”, Nature Nanotechnology , Vol 6, (2011) are all incorporated herein by reference.

The present invention provides a sequencing device using graphene nanoribbons. The device includes a substrate on which nano grooves are formed, a graphene nanoribbon positioned on the substrate, a current application electrode connected to both ends of the graphene nanoribbon, and a gate voltage application electrode connected to the bottom of the substrate.

The graphene nanoribbons have a two-dimensional structure of a single layer made of carbon. Graphene nanoribbons change their electrical properties to conductors, semiconductors, and non-conductors. The semiconducting properties of the graphene nanoribbons formed in the chair-like direction represent semiconducting properties, and the decrease in conductivity is most pronounced in the semiconducting graphene nanoribbons. The analysis device of the present invention is designed to measure the decrease in conductivity at a specific gate voltage, and therefore, the graphene nanoribbons are preferably formed in a chair shape direction.

The graphene nanoribbons preferably have a width of 10 nm or less. The wider the width, the greater the number of bases attached to the graphene nanoribbons, making it difficult to separate the signal from each base. Therefore, the graphene nanoribbons should be ideally around 1nm wide, with only one base attached.

The graphene nanoribbons are preferably located across the nanogrooves, that is, orthogonal to the nanogrooves. It is preferable that both ends of the graphene nanoribbons are fixed to the substrate through an electrode.

The nano grooves serve as a passage through which DNA to be analyzed passes, and are preferably formed in a straight line. The width of the nano grooves is preferably such that a single helix DNA can pass tightly, ideally about 1 nm. If the width is too wide, the base can pass through the graphene nanoribbon without forming a stacked structure, or with DNA strands.

Using the sequencing device, the present invention provides a method for analyzing the nucleotide sequence of DNA. The method includes applying a bias voltage between both electrodes (source and drain electrodes) of the graphene nanoribbons and applying an alternating gate voltage to the gate electrode, and the DNA to be analyzed to pass under the graphene nanoribbons through the nanogrooves. Towing, detecting in real time the current flowing through the graphene nanoribbons, and analyzing the relationship between the current and the gate voltage of the graphene nanoribbons over time.

In the assay device, the DNA base forms a stacked structure due to strong aromatic interaction with the graphene nanoribbons as it passes under the graphene nanoribbons. Therefore, it is possible to minimize the fluctuation of the base generated when using the conventional nanopores. In addition, the graphene nanoribbon current of a constant distribution can be measured according to the base passing through the graphene nanoribbon. After measuring the graphene nanoribbon current depending on a specific range of gate voltages, the new data processing method developed in the present invention can analyze the measured current to reliably decode the genetic information within a few hours.

The faster the change cycle is, the better the AC gate voltage is. That way, less structure changes in one measurement and statistically more accurate data can be obtained. The gate voltage is preferably applied at a speed of at least MHz, and the range is preferably between about -2V and about 2V.

Towing DNA so that the DNA to be analyzed passes under the graphene nanoribbons can be performed by an external electric field. Since the DNA is negatively charged, the DNA will move in the opposite direction of the electric field. In addition to the electric field, for example, the flow of the DNA aqueous solution can be controlled to allow DNA to pass under the nanoribbons.

In one aspect of the invention, there is provided a method for sequencing a base sequence using an analysis element. The analysis device includes a graphene nanoribbon disposed in a direction crossing the nanochannel on a substrate on which a nanochannel is formed, a gate electrode is provided below the substrate, and a source electrode and a drain are provided at both ends of the graphene nanoribbon. Each electrode is provided. The method includes applying a bias current between the source and drain electrodes and applying a gate voltage to the gate electrode, passing a single helix DNA through the nanochannel under the graphene nanoribbon, and Detecting a change in current of the graphene nanoribbon that occurs when the base of the DNA interacts with the graphene nanoribbon in real time; and analyzing the gate voltage and the graphene nanoribbon current over time. .

The detected current is converted into a two-dimensional distribution of energy over time through real-time histogram analysis. From this conversion result, a base signal at a specific energy can be extracted, and information of a single base can be obtained from the extracted signal. The time point at which each base signal starts and ends can be determined through a two-dimensional autocorrelation function.

In one aspect, the present invention is characterized by analyzing the DNA sequence by measuring the current change of the graphene nanoribbon when passing the DNA down the graphene nanoribbon in the method for analyzing the base sequence of the DNA.

A sequencing device comprising a sequencing device according to the invention and a means for passing DNA through the analysis device can be constructed.

The device and method of the present invention can quickly decode genetic information as quickly as possible within a few hours. In addition, the technique according to the present invention, unlike the conventional method, there is no cutting-amplification-labeling process, so it is possible to decode genetic information at low cost since no large-scale equipment is required. Therefore, the present invention can be applied to a wide variety of fields in the future bio fields, such as personal medicine, gene therapy, cancer therapy.

1A is an exemplary diagram of a genetic information decoding device according to the present invention.
FIG. 1B shows the stacked structure through aromatic interaction between DNA base and graphene nanoribbon, as seen in molecular dynamics computer simulations.
Figure 2a is a graph showing the permeability function of the graphene nanoribbons shown when each of the four different bases attached, and the graphene nanoribbons for comparison.
FIG. 2B shows a molecular orbital picture when no pano resonance occurs, and FIG. 2C shows a molecular orbital picture when a pano resonance occurs.
3 is a diagram illustrating the effect of real-time histogram processing used in the present invention from the transmittance data.
4A to 4E show real-time histograms obtained from 5'-GCATCGCT-3 'genes, extracted signals, single base probabilities, two-dimensional autocorrelation functions, and sequence completion, respectively.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. In the following description of the present invention, if it is determined that a detailed description of a known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted. The following terms are defined in consideration of the functions of the present invention, and may be changed according to the intentions or customs of the user, the operator, and the like. Therefore, the definition should be based on the contents throughout this specification.

The genetic information decoding element according to the embodiment of the present invention is configured as shown in FIG. The readout device has a graphene nanoribbon (GNR) mounted on the nanochannel, and provided with three electrodes of a source (S), a gate (G), and a drain (D).

The nanochannel, which is the passage through which DNA passes, is preferably formed on a substrate made of silicon nitride. As long as it does not interact strongly with the DNA base, substrates of other materials besides silicon nitride may be used.

The graphene nanoribbon is a strip having a narrow, long edge of a single atom-thick sheet made of carbon, and according to the present invention, a graphene nanoribbon of several nanometers width, as shown in FIG. It is located on the silicon nitride substrate in the direction across the. Below the nanochannel of the substrate is provided a gate electrode E _G for applying a gate voltage. Opposite ends of the nanoribbons are provided with electrodes for applying a current to the nanoribbons, that is, a source electrode E _S and a drain electrode E _D , respectively.

The dielectric information decoding device is an electronic device for analyzing a current flowing through the graphene nanoribbons. The basic operating principle of the genetic information decoding device is as follows.

Preferably, single helical DNA dissolved in a solvent, which is water, is attracted to the graphene nanoribbons by an external electric field, and each DNA base forms a stable stacked structure through aromatic interaction with the graphene nanoribbons. At this time, a bias voltage is applied to the electrodes E _S and E _D of both graphene nanoribbons, and an AC gate voltage V _G is applied to the gate electrode E _G under the silicon nitride substrate, thereby providing graphene. The current flowing through the nanoribbon (I _GNR ) is measured in real time. DNA sequences are analyzed based on three-dimensional graphs of I _GNR -V _G -time measured in real time.

For the validity of the above principle of operation, how the structure of the DNA base forms a stable stacked structure under the graphene nanoribbons, how the electrical conductivity changes when different DNA bases are attached to the graphene nanoribbons, It is described below whether genetic information can be decoded by analyzing the I _GNR -V _G -time signal.

In the present invention, a strong aromatic interaction between the DNA base and the graphene nanoribbons is used to form a stable molecular structure at the nano level. This overcomes the limitations of conventional methods using nanopores.

DNA bases are largely divided into purine series of adenine (A) and guanine (G), and pyrimidine series of cytosine (C) and thymine (T). Both purines and pyrimidines have aromatic properties, which show large binding energies (~ 20 kcal / mol) due to π-π interaction with graphene nanoribbons with delocalized π orbitals. Form a stable structure with the ribbon. Since these π-π interactions provide much stronger bonds than the π-H interactions between water molecules and graphene nanoribbons, DNA bases in aqueous solutions are substantially unaffected by the solvent effect, making them stable with graphene nanoribbons. The structure can be formed. This fact can be confirmed through the results of the molecular dynamics simulation experiment of FIG. 1B using the CHARMM parameter and the NAMD program package based on the schematic diagram of the DNA genetic information decoding device shown in FIG. 1A.

Pulling with a constant force of, for example, 35 kcal / mol / angstroms to create an electric field in a single helix DNA, the DNA moves towards the graphene nanoribbon and the DNA base passes underneath the graphene nanoribbon, as shown in FIG. 1B. In addition, π-π interactions form stable stacked structures. From this, it can be seen that even at about 300K at room temperature, it forms a stable stacked structure between the DNA base in the aqueous solution and the graphene nanoribbons.

The present invention takes advantage of the difference in the I _GNR -V _G graph that appears when different DNA bases interact with graphene nanoribbons. The current (I _GNR ) flowing through the graphene nanoribbons can be known through T (E), a transmittance function based on a non-equilibrium Green's function, as shown in Equation (1).

(1)

(One)

In Equation (1), Σ is self-energy and expresses the effect of the electrode, and L and R represent the left electrode and the right electrode, respectively. When a small bias voltage is applied, the magnitude of the current (I _GNR ) that can be obtained using the Landauer-Buttiker equation is proportional to the transmittance, and the gate voltage (V _G ) corresponds to the energy EE _F (where E _F is Fermi Energy). As can be seen from Figure 2a, the present invention utilizes the resonance energy level in the transmittance depends on the type of DNA base. This change in permeability is due to the Fano resonance caused by the interaction between the DNA base and the graphene nanoribbons. This can be seen that the wave function of electrons flowing through the graphene nanoribbons cause a destructive interference with the wave function of electrons passing through the DNA base, and the permeability of electrons is rapidly dropped at the molecular orbital energy level of the DNA base. 2B and 2C show energy levels at which phono resonance does not occur (EE _F = -0.9 eV) and energy levels at which phono resonance occurs (EE _F = -1.4 eV) when cytosine bases interact with graphene nanoribbons. Shows molecular orbitals at. When pano resonance occurs, the electron cloud is bound to the DNA base so that electrons do not flow through the graphene nanoribbons.

Based on the previous molecular dynamics simulations and permeability calculations, we describe how real devices decode DNA sequences. Molecular dynamics simulations determine the structure of DNA and graphene nanoribbons in real time. Based on the determined structure, the transmittance function is calculated for each time. By processing the data obtained from the permeability function through the data processing method newly developed by the present inventors, DNA sequencing can be analyzed. A more detailed description of data processing follows.

Due to the discontinuity of the measurement method due to the discontinuous gate voltage application, the transmittance function shows only scattered values. Therefore, the permeability function alone cannot easily determine the type of DNA base. Therefore, the present invention uses a new statistical method called real-time histogram. The method is as following formula (2).

(2)

(2)

Where D (E, t) is the histogram of the peak position at time t. As can be seen from Equation (2), the histogram at the current time is created based on the data of the previous specific time interval (Δ). Using this method, the scattered data has a constant distribution, which depends on the type of DNA base. 3 shows that the scattered transmittance data forms a two-dimensional distribution through real-time histogram analysis.

Figure 4a shows a real-time histogram obtained according to the invention of the permeation peak position when 5'-GCATCGCT-3 'DNA passes through the sequencing device designed as in Figure 1. For each base, the histogram is distributed around a specific energy. Where red represents the maximum value and blue represents the minimum value. From the present invention, the distribution of cytosine (C) is shown at EE _F = 1.7 eV, guanine (G) at EE _F = -0.7 eV, adenine (A) and cytosine (C) at -1.2 eV, and guanine at -1.5 eV. It can be seen that (G) and thymine (T) appear simultaneously. Extracting the base signal (S _BASE ) at each energy is as shown in Figure 4b.

From the signals described above, four types of bases can be clearly separated using projection / subtraction methods. Equation (3), a probability function designed for each base, shows how to extract information from a single base.

(3)

(3)

The signal of a single base obtained through Equation (3) can be accurately separated over time as shown in Figure 4c. At this time, the signal for the cytosine, the seventh base in Figure 4c is not seen because the cytosine did not adhere to the graphene nanoribbons. This uncertainty appears only in cytosine and can be resolved through parallel experiments.

In the present invention, a newly developed two-dimensional autocorrelation function is introduced to determine the starting point and ending point of the base signal. Determining when a single nucleotide signal appears is very important in automating DNA sequencing devices using nanodevices. The two-dimensional autocorrelation function analysis is described in more detail as follows.

The two-dimensional autocorrelation function follows the following equation (4).

(4)

(4)

Equation (4) adds a time dependency to the normal autocorrelation function. Since the sequencing method needs to process dynamic information that changes with time, it processes information in a specific section τ rather than infinite time. J (E, t) means the event function at time t, which corresponds to the transmittance function at time t. Since the dot product of the permeability function at times t and t + t 'is close to 1 for the same base and close to 0 for different bases, C (t, t ₀ ; τ) becomes an inverse triangle as new bases are introduced. The pattern of the shape appears. 4D shows the two-dimensional autocorrelation function for 5′-GCATCGCT-3 ′ DNA. Here, the dashed black line means the part where the base ends, and the dashed white line means the part where the base starts. Based on FIGS. 4C and 4D, the complete sequence of FIG. 4E can be obtained.

Although the present invention has been described with reference to specific exemplary embodiments, the present invention is not limited by the embodiments, but only by the claims. It should be appreciated that those skilled in the art can change or modify the embodiments without departing from the scope and spirit of the present invention.

Claims (20)

A sequencing device using graphene nanoribbons,
A nano groove formed substrate;
Graphene nanoribbons positioned on the substrate;
A current application electrode connected to both ends of the graphene nanoribbons; And
A gate voltage application electrode connected under the substrate;
A sequencing element comprising a. The nucleotide sequence analysis device of claim 1, wherein the graphene nanoribbons have a single layered two-dimensional structure made of carbon, and are formed in a chair shape direction. The nucleotide sequence analysis device of claim 1, wherein the graphene nanoribbons have a width of 10 nm or less. According to claim 1, wherein the graphene nanoribbon sequence analysis device, characterized in that across the nano grooves of the substrate. The sequencing device of claim 1, wherein the nano grooves of the substrate are formed in a straight line and serve as a passage through which DNA passes. A method of analyzing a nucleotide sequence using the nucleotide sequence analyzing device according to any one of claims 1 to 5,
Applying a bias voltage between both electrodes of the graphene nanoribbons and applying an alternating gate voltage to the gate electrode;
Towing the DNA to be analyzed to pass under the graphene nanoribbons through the nanogrooves;
Detecting a current flowing through the graphene nanoribbon in real time; And
Analyzing the relationship between the current and the gate voltage of the graphene nanoribbon with respect to time;
Sequence analysis method comprising a. The sequencing method according to claim 6, wherein the gate voltage is applied at a speed of a specific range of voltages at a level of MHz or higher. The method of claim 6, wherein the traction of the DNA is performed by an external electric field. The method of claim 6, wherein the DNA is provided in the form of an aqueous solution at room temperature. The sequencing method of claim 6, wherein the current flowing through the graphene nanoribbons is obtained through a permeability function based on an unbalanced green function of Equation (1):

(One). The sequencing method of claim 10, wherein the analyzing comprises converting the data obtained by Equation (1) into a real-time histogram through Equation (2):

(2). The method of claim 11, wherein the analyzing comprises extracting a base signal related to a specific energy from the real-time histogram. The method of claim 12, wherein the analyzing comprises projecting and removing the extracted base signal through Formula (3) to obtain information of a single base.

(3). The nucleotide sequence analysis method of claim 13, comprising determining a start point and an end point of a specific base signal through a two-dimensional autocorrelation function of Equation (4):

(4). A sequencing method using a sequencing element as an analyzing element, the analyzing element includes a graphene nanoribbon disposed in a direction crossing the nanochannel on a substrate on which the nanochannel is formed, and a gate electrode is provided below the substrate. Both ends of the graphene nanoribbon are provided with a source electrode and a drain electrode, respectively.
Applying a bias current between the source and drain electrodes and applying a gate voltage to the gate electrode;
Passing single helix DNA through the nanochannels under the graphene nanoribbons;
Detecting a change in current of the graphene nanoribbon when the base of the DNA interacts with the graphene nanoribbon; And
Analyzing the gate voltage and the graphene nanoribbon current over time;
Sequence analysis method comprising a. The method of claim 15, wherein the detected current is converted into a two-dimensional distribution of energy over time through a real-time histogram analysis. The nucleotide sequence analysis method of claim 16, wherein a base signal at a specific energy is extracted from the conversion result of the real-time histogram analysis, and information of a single base is obtained from the extracted signal. 18. The method of claim 17, wherein the starting point and ending point of each base signal are determined through a two-dimensional autocorrelation function. A sequencing device according to any one of claims 1 to 5, and a sequencing device comprising means for passing DNA through the analysis device. delete

Images