KR101267789B1 - DNA analyzing apparatus using nanopore structure, analyzing method and apparatus for quantitative detection of PCR products - Google Patents

Abstract

Provided are a device for DNA analysis, a DNA analysis method, and a PCR quantitative detection device using a nanopore structure. DNA analysis apparatus using a nano-pore structure according to an embodiment of the present invention, the chamber containing a solution, the first region and the second region; A first electrode positioned in the first region; A second electrode positioned in the second region opposite the first electrode; And a nano pore film disposed between the first electrode and the second electrode, the nano pore film including a nano pore penetrating the conductive layer and the conductive layer. An electrical signal portion electrically connected to the conductive layer, the first electrode, and the second electrode, for applying a first electrical signal and receiving a second electrical signal therefrom; It is characterized by detecting DNA.

Description

DNA analyzing apparatus using nanopore structure, analyzing method and apparatus for quantitative detection of PCR products {DNA analysis apparatus using nanopore structure, analyzing method and apparatus for quantitative detection of PCR products}

The present invention relates to an apparatus for DNA analysis using a nano-pore structure, an analytical method and a PCR quantitative detection apparatus using the same, and more particularly, an apparatus for DNA analysis using nano-pores capable of analyzing DNA without labeling. It relates to an analysis method and a PCR quantitative detection device using the same.

Research has been actively conducted to control the flow of charged particles in a solvent by selectively controlling the movement of ions when a cation and an anion are present in the solvent. In recent years, many researches are being conducted in accordance with the global research direction to determine the principle of biological mechanisms by regulating the flow of biological materials such as DNA, RNA and protein. In order to manufacture such a device, it is required to form a nanometer-sized channel structure or a pore structure.

It is an object of the present invention to provide an apparatus for DNA analysis and a PCR quantitative detection apparatus that can easily analyze DNA by detecting an electrical signal using a nanopore structure.

Another object of the present invention is to provide a method for easily analyzing DNA by applying and detecting an electrical signal using a DNA analysis apparatus.

An apparatus for DNA analysis using a nanopore structure according to an embodiment of the present invention is provided. The device for DNA analysis using the nano-pore structure includes a chamber containing a solution and including a first region and a second region; A first electrode positioned in the first region; A second electrode positioned in the second region opposite the first electrode; And a nano pore film disposed between the first electrode and the second electrode and including a conductive layer and a nano pore passing through the conductive layer. And an electrical signal unit electrically connected to the conductive layer, the first electrode, and the second electrode to apply a first electrical signal and receive a second electrical signal therefrom. It is characterized by detecting the DNA in the solution.

In some embodiments of the present invention, the second electrical signal that changes when the DNA passes through the nanopores may be used to detect one or more bases constituting the DNA.

In some embodiments of the present invention, the DNA may be sequentially sequenced by detecting the second electrical signal having a different value depending on the base constituting the DNA.

In some embodiments of the present invention, the nano-pore film may further include a first insulating layer stacked on the upper side, the lower side, or both sides of the conductive layer.

In some embodiments of the present invention, the nano-pore film may include a second insulating layer formed on a side of the conductive layer adjacent to the nano-pore and a side of the first insulating layer and surrounding the first insulating layer. It may further include.

In some embodiments of the present invention, the conductive layer includes titanium nitride (TiN), the first insulating layer includes silicon oxide (SiO ₂ ), and the second insulating layer is titanium oxide (TiO _2). ) May be included.

In some embodiments of the present disclosure, the DNA analysis device may operate as an ion field transistor by a voltage applied by the electrical signal unit to the conductive layer, the first electrode, and the second electrode.

In some embodiments of the present invention, the DNA analysis device, the nano-pores may have a cylindrical shape of the same diameter of the upper and lower sides or a truncated conical shape of the diameter of the upper and lower sides.

A DNA analysis method using a device for DNA analysis using a nano-pore structure according to an embodiment of the present invention is provided. DNA analysis method using the nano-pore structure, preparing the DNA analysis apparatus; Receiving a solution containing DNA in the chamber of the DNA analysis device; Applying a first electrical signal to the conductive layer, the first electrode, and the second electrode from the electrical signal portion; And receiving the second electrical signal generated when the DNA passes through the nanopores.

In some embodiments of the present disclosure, the receiving of the second electrical signal may include changing and applying the first electrical signal applied to the conductive layer in order to control the moving speed of the DNA. It may further include.

In some embodiments of the present invention, as the first electrical signal applied to the conductive layer is increased, the moving speed of the DNA may be reduced.

Provided is a PCR quantitative detection apparatus using a nano-pore structure according to an embodiment of the present invention. PCR quantitative detection apparatus using the nano-pore structure at least one input unit for providing a sample and a buffer solution containing DNA; A reaction unit in which amplification of the DNA is performed; And a DNA analyzing apparatus according to any one of claims 1 to 8, and comprising a detection unit for quantitatively analyzing the DNA through an electrical signal generated while the amplified DNA passes through the nanopores. .

In some embodiments of the present disclosure, a concentration controller may be further disposed between the reaction unit and the detection unit to control the concentration of the amplified DNA by receiving the buffer solution from the input unit.

According to the apparatus for DNA analysis using the nano-pore structure of the present invention, by using a nano-pore having a size of less than 10nm and can easily control the charge on the surface of the nano-pores, it is possible to efficiently control the movement speed of DNA. As a result, time for DNA analysis can be secured and the magnitude of the electrical signal can be adjusted.

In addition, according to the DNA analysis method using the nano-pore structure of the present invention, it is possible to efficiently sequence the DNA by controlling the movement of the DNA through the regulation of the voltage applied to the membrane on which the nano-pores are formed.

1 is a perspective view showing a device for DNA analysis using a nano-pore structure according to an embodiment of the present invention.
2 is a perspective view showing the structure of a nano-pore membrane according to the present invention.
3A-3E are cross-sectional views shown in order of process to illustrate an exemplary method for producing nanopore membranes in accordance with the present invention.
4A and 4B are photographs showing a state in which nanopores are formed according to the present invention.
5 is a graph showing ion conductance according to the concentration of potassium chloride (KCl) solution measured in the nano-pore structure of the DNA analysis device according to the present invention.
6A and 6B are graphs showing ion currents according to a drain voltage V _D and a gate voltage V _G of an ion transistor of a device for DNA analysis according to the present invention.
FIG. 7A is a schematic diagram showing DNA passing through the nanopores according to the present invention, and FIG. 7B is a graph showing the flow of electric current to explain the method of sequencing DNA.
8 is a flowchart illustrating a DNA analysis process using a device for DNA analysis using the nanopore structure according to the present invention.
9 is a schematic diagram showing a PCR quantitative detection apparatus using a nano-pore structure according to an embodiment of the present invention.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The embodiments of the present invention are described in order to more fully explain the present invention to those skilled in the art, and the following embodiments may be modified into various other forms, It is not limited to the embodiment. Rather, these embodiments are provided so that this disclosure will be more faithful and complete, and will fully convey the scope of the invention to those skilled in the art.

In the following description, when a layer is described as being on top of another layer, it may be directly on top of the other layer, with a third layer intervening therebetween. In the drawings, the thickness and size of each layer are exaggerated for convenience and clarity of description, and the same reference numerals refer to the same elements in the drawings.

Although the terms first, second, etc. are used herein to describe various elements, components, regions, layers and / or portions, these members, components, regions, layers and / It is obvious that no. These terms are only used to distinguish one member, component, region, layer or section from another region, layer or section. Thus, a first member, component, region, layer or section described below may refer to a second member, component, region, layer or section without departing from the teachings of the present invention.

Hereinafter, embodiments of the present invention will be described with reference to the drawings schematically showing ideal embodiments of the present invention. In the figures, for example, variations in the shape shown may be expected, depending on manufacturing techniques and / or tolerances. Accordingly, embodiments of the present invention should not be construed as limited to any particular shape of the regions illustrated herein, including, for example, variations in shape resulting from manufacturing.

1 is a perspective view showing an embodiment of a device for DNA analysis 100 using a nano-pore structure according to the technical idea of the present invention.

Referring to FIG. 1, the apparatus 100 for DNA analysis includes a membrane (hereinafter, referred to as a “nanopore membrane”) 20 in which a chamber 10 and nanopores 25 are formed. The first region 12 and the second region 14 of the chamber 10 are disposed on both sides of the nanopore film 20. The first electrode 30 and the second electrode 40 are disposed in the first region 12 and the second region 14, respectively. The nano pore membrane 20 includes a nano pore 25, which may be located at the center of the nano pore membrane 20, for example. The nano-pore film 20 may include a conductive layer 21, first insulating layers 22a and 22b on both sides of the conductive layer 21, and side surfaces of the conductive layer 21 adjacent to the nano-pores 25. The second insulating layer 24 is formed on the side surfaces of the first insulating layers 22a and 22b and surrounds the first insulating layers 22a and 22b. The electrical signal unit 50 that can transmit and receive electrical signals is disposed outside the chamber 10.

The chamber 10 may be separated into the first region 12 and the second region 14 by the nano-pore membrane 20, and may be configured as separate chambers, respectively. The chamber 10 is for containing a solution containing DNA, and the first electrode 30 and the second electrode 40 in each of the first region 12 and the second region 14 of the chamber 10. Place it. The chamber 10 may be made of one or more materials of glass, polydimethylsiloxane (PDMS), and plastic. The solution contained in the chamber 10 is a solution containing DNA, and is prepared in a fluid state by dissolving DNA in a conductive solvent, and any conductive solvent may be used. The subject of analysis is not limited to DNA, but may be DNA, RNA, peptide or protein. The solution may be an electrolyte solvent such as hydrochloric acid (HCl), sodium chloride (NaCl) or potassium chloride (KCl). Potassium chloride (KCl) is characterized by almost no difference in the ion mobility (mobility) of the cation and anion. The apparatus may further include an injection unit (not shown) and a discharge unit (not shown) capable of injecting and discharging the solution into the chamber 10 outside the chamber 10. The chamber 10 may have a small capacity, and the length in either direction may have dimensions of several micrometers.

The first electrode 30 may be disposed in the first region 12 of the chamber 10, and the second electrode 40 may be disposed in the second region 14 of the chamber 10. The first electrode 30 and the second electrode 40 may apply a voltage to the solution in the chamber 10 to flow ions in the solution, resulting in the flow of current. The first electrode 30 and the second electrode 40 are aluminum (Al), gold (Au), beryllium (Be), bismuth (Bi), cobalt (Co), hafnium (Hf), indium (In), and manganese. (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), palladium (Pd), platinum (Pt), rhodium (Rh), rhenium (Re), ruthenium (Ru), tantalum (Ta), tellium (Te), titanium (Ti), tungsten (W), zinc (Zn), zirconium (Zr), nitrides thereof, and silicides thereof. The first electrode 30 and the second electrode 40 may each be a single layer or a composite layer. For example, the first electrode 30 and the second electrode 40 may be a composite layer of silver (Ag) or silver chloride (AgCl). The first electrode 30 and the second electrode 40 may be configured to include the same material or different materials. In addition, the first electrode 30 and the second electrode 40 may be disposed to be close to the nano-pore film 20.

The nano-pore film 20 may include a conductive layer 21, first insulating layers 22a and 22b on both sides of the conductive layer 21, and side surfaces of the conductive layer 21 adjacent to the nano-pores 25. The second insulating layer 24 is formed on the side surfaces of the first insulating layers 22a and 22b and surrounds the first insulating layers 22a and 22b. The nano pore membrane 20 includes a nano pore 25 formed at a central portion, and the nano pore 25 penetrates through the nano pore membrane 20. The nano-pore membrane 20 will be described in detail below with reference to FIG. 2.

The electrical signal unit 50 may apply a first electrical signal, for example, a voltage, to the conductive layer 21 of the first electrode 30, the second electrode 40, and the nanopore film 20. In addition, the electrical signal unit 50 may receive a second electrical signal, for example, a current, from the conductive layer 21 of the first electrode 30, the second electrode 40, and the nanopore film 20. .

The voltage applied to the conductive layer 21 of the nanopore film 20 is a gate voltage (V _G ), the voltage applied to the first electrode 30 is a source voltage (V _S ), the second electrode 40 ) May be referred to as a drain voltage (V _D ). The electrical signal part 100b may be electrically connected to the conductive layer 21, the first electrode 30, and the second electrode 40 of the nanopore film 20 through a conductive member, for example, a conductive wire. In particular, the portion connected to the conductive layer 21 may be connected by a probe (not shown).

By the gate voltage (V _G ), the source voltage (V _S ) and the drain voltage (V _D ) applied by the electrical signal unit 50, the device for DNA analysis 100 is an ionic field effect transistor (IFET). Can be operated as Ion field transistors are similar in principle to conventional semiconductor field effect transistors except that the carriers traveling through the channel are electrolyte ions, not electrons or holes. Therefore, the ion current flows due to the movement of the ions, not the movement of the electrons, and the nanopores 25 serve as channels for the movement of the ions. When receiving the electrolyte solution in the chamber 10, the ionized cations and anions can be moved in either direction by the source voltage (V _S ) and the drain voltage (V _D ) applied to the chamber 10, The on state and the off state of the transistor may be controlled by the gate voltage V _G applied to the conductive layer 21 of the nanopore film 20. When DNA is included in the solution, the behavior of the DNA according to the electrical signal is described in detail below with reference to FIGS. 7A and 7B.

2 is a perspective view showing the structure of the nano-pore membrane 20 according to the present invention.

Referring to FIG. 2, the nano pore film 20 may include a conductive layer 21, first insulating layers 22a and 22b on both sides of the conductive layer 21, and the conductive layer adjacent to the nanopore 25. 21 and a second insulating layer 24 formed on the side surfaces of the first insulating layers 22a and 22b and surrounding the first insulating layers 22a and 22b. That is, the periphery of the nano pore 25 is a form in which the second insulating layer 24 extends to surround the conductive layer 21 and the first insulating layers 22a and 22b, and the second periphery of the nano pore 25. The insulating layer 24 may serve as a gate insulating film of the ion transistor. The nano pores 25 may have a cylindrical shape having the same upper and lower diameters or a truncated conical shape having different upper and lower diameters. The thickness of the conductive layer 21, the first insulating layers 22a and 22b, and the second insulating layer 24 constituting the nanopore film 20 may be several tens of nanometers. 21) may have a thickness of 20 nm to 40 nm, for example about 30 nm, and the first insulating layers 22a and 22b may each have a thickness of 15 nm to 25 nm, for example about 20 nm. The second insulating layer 24 may have a thickness of 20 nm to 40 nm.

The conductive layer 21 includes aluminum (Al), gold (Au), beryllium (Be), bismuth (Bi), cobalt (Co), hafnium (Hf), indium (In), manganese (Mn), and molybdenum (Mo). , Nickel (Ni), lead (Pb), palladium (Pd), platinum (Pt), rhodium (Rh), rhenium (Re), ruthenium (Ru), tantalum (Ta), tellurium (Te), titanium (Ti) , Tungsten (W), zinc (Zn), zirconium (Zr), nitrides thereof, and silicides thereof. The conductive layer 21 may be a single layer or a composite layer.

The first insulating layers 22a and 22b and the second insulating layer 24 may be formed of silicon oxide (SiO ₂ ), silicon nitride (Si ₃ N ₄ ), silicon oxynitride (SiON), or high-k dielectric constant (high-k). It may include any one or more of the dielectric layer. The first insulating layers 22a and 22b and the second insulating layer 24 may be a single layer or a composite layer. The high-k dielectric layer includes aluminum oxide (Al ₂ O ₃ ), tantalum oxide (Ta ₂ O ₃ ), titanium oxide (TiO ₂ ), yttrium oxide (Y ₂ O ₃ ), zirconium oxide (ZrO ₂₎ ), Zirconium silicon oxide (ZrSi _x O _y ), hafnium oxide (HfO ₂ ), hafnium silicon oxide (HfSi _x O _y ), lanthanum oxide (La ₂ O ₃ ), lanthanum aluminum oxide (LaAl _x O _y ), lanthanum hafnium At least one of an oxide (LaHf _x O _y ), a hafnium aluminum oxide (HfAl _x O _y ), and a praseodymium oxide (Pr ₂ O ₃ ) may be included.

3A to 3E are cross-sectional views according to a process sequence to explain an exemplary method for manufacturing a nano-pore film according to the spirit of the present invention.

Referring to FIG. 3A, a laminated film 20a in which the first insulating layer 22b, the conductive layer 21, and the second insulating layer 22a are sequentially stacked is formed on the substrate 60, and the first insulating layer 22b is formed on the substrate 60. 1 mask layer 70 is laminated. The films may be deposited using a deposition method such as chemical vapor deposition (CVD) or reactive sputtering. Subsequently, second mask layers 80a and 80b are stacked on the lower surface of the substrate 60 and the first mask layer 70. The substrate 60 may be, for example, a silicon (Si) substrate. The first mask layer 70 and the second mask layers 80a and 80b may include oxides, nitrides, and oxynitrides. The first mask layer 70 and the second mask layers 80a and 80b may include a material having different etching selectivity. For example, the first mask layer 70 may include silicon oxide (SiO ₂ ), and the second mask layers 80a and 80b may include silicon nitride (Si ₃ N ₄ ). Or vice versa.

Referring to FIG. 3B, the step of etching the substrate 60 is performed. A pattern is formed in the second mask layer 80b on the lower surface of the substrate 60. A pattern may be formed using a separate mask layer (not shown), and the pattern may be formed by etching the second mask layer 80b. The etching may use reactive ion etching (RIE). Next, the substrate 60 is etched using the patterned second mask layer 80b. The substrate 60 may be etched using anisotropic wet etching using potassium hydroxide (KOH). By etching the central portion of the substrate 60 in this step, the formation of the openings 25a (see FIG. 3D) in a later process may be facilitated.

Referring to FIG. 3C, after the photoresist layer 90 is stacked on the second mask layer 80a, a pattern for forming nanopores is formed by using electron beam lithography. The photoresist layer 90 may include poly methyl methacrylate (PMMA) and may be applied by spin-coating. Using the pattern, the second mask layer 80a is etched to remove the photoresist layer 90.

Referring to FIG. 3D, a two-step etching process of forming the opening 25a in the center of the stacked film 20a is performed. First, the first mask layer 70 is etched using the second mask layer 80a on which the pattern is formed. The etching may use RIE. Next, the laminated film 20a is etched using the patterned first mask layer 70. Thereby, the opening part 25a is formed in the center of the laminated film 20a. The etching may use RIE, and the opening 25a may be formed using a focused ion beam (FIB). The opening 25a may have a diameter of 100 nm or less. The laminate layer 20a may remove the substrate 60, the first mask layer 70, and the second mask layers 80a and 80b existing in the peripheral portion of the lower portion through an additional etching process, and the laminate layer 20a may be removed. It may be used to manufacture the device in a state including the remaining portion of the lower substrate 60 and the second mask layer 80b.

 Referring to FIG. 3E, the second insulating layer 24 may be deposited to form the nanopore film 20 of FIG. 2. Atomic layer deposition (ALD) may be used as the deposition method of the second insulating layer 24. When ALD is used as compared with the case of using CVD, a film of several nanometers in thickness can be uniformly grown, thereby allowing a uniform film to be grown around the opening 25a (see FIG. 3D). As the second insulating material is deposited around the opening 25a, the circumference of the opening 25a becomes smaller and smaller, and the gas molecules serving as the source are no longer allowed to enter the opening 25a so that the deposition cannot be continued. Done. This is called a self-limiting effect, whereby a nano pore 25 having a uniform size can be formed. The deposition thickness of the second insulating layer 24 is controlled according to the size of the desired nano-pore 25, thereby forming a nano-pore 25 having a size of 10nm or less.

4A and 4B are photographs showing how the nanopores 25 are formed.

In conjunction with FIG. 2, reference is made to FIGS. 4A and 4B. 4A is a photograph of the opening 25a before the second insulating layer 24 is deposited, and FIG. 4B is a photograph of the nano pores 25 after the second insulating layer 24 is deposited.

FIG. 4A is a scanning electron microscopy (SEM) micrograph showing an opening 25a formed using electron beam lithography and reactive ion etching, wherein the opening 25a is about 70 to 80 nm in size. 4B is a scanning electron micrograph showing a state in which nanopores 25 of 10 nm or less are formed after deposition of the second insulating layer 24. By the above-described self-limiting effect, the nanopores 25 having a diameter reduced by about 60 nm to 70 nm may be formed from the opening 25a.

5 is a graph showing ion conductance according to the concentration of potassium chloride (KCl) solution measured in the nano-pore structure of the device for DNA analysis according to the technical idea of the present invention.

Referring to FIG. 5, when the concentration of potassium chloride (KCl) is high, the ionic conductance is linearly proportional to the electrolyte concentration. The nano-pore film 20 used for the measurement is the electrode layer 21 of 30 nm titanium nitride (TiN), the first insulating layers 22a and 22b of 20 nm silicon nitride (Si ₃ N ₄ ), and by ALD It is a nano-pore film 20 in which titanium oxide (TiO ₂ ), which is the second insulating layer 24, is deposited to form nano-pores 25 of 10 nm or less. When the concentration of potassium chloride (KCl) is lowered, the ion conductance does not have a linear proportional relationship as described above, and exhibits a specific conductance.

This conductance characteristic is that the second insulating layer 24 itself has a surface charge, and an electrical double layer 26 composed of counter ions is formed around the second insulating layer 24 so that the second insulating layer is formed. This is because screening the surface charge of (24). The surface charge of the second insulating layer 24, which is a gate insulating film, may vary depending on an insulating material. For example, silicon oxide (SiO ₂ ), titanium oxide (TiO ₂ ), and tin oxide (SnO ₂ ) may have surface charges. Negative, aluminum oxide (Al ₂ O ₃ ) and zinc oxide (ZnO) may have a positive surface charge. The phenomenon of masking the surface charge as described above can be interpreted by Debye leghth, which is inversely proportional to the square root of the concentration of the electrolyte in the solution.

Referring to the illustration of the nano-pore film 20 inserted in the graph of FIG. 5, at a low concentration, a relatively thick electrical bilayer 26 is formed. The electrical double layer 26 overlaps in the nanopores 25 so that the interior of the nanopores 25 is filled with counter ions, and the type and amount of ions present in the nanopores 25 are potassium chloride (KCl). It does not change according to the concentration of the concentration range where the saturation of the ion conductance occurs. This ion selectivity is the basis for operating as an ion transistor. As the size of the nano-pores 25 becomes smaller, the electrical double layer 26 may overlap in the nano-pores 25 even if the draw length is small. Thus, even if a higher concentration of solution is used, the nanopores 25 can have ion selectivity. In the case of using a high concentration of solution, the high ion concentration also increases the measured ion current, thereby increasing the accuracy of the measurement.

6A and 6B are graphs illustrating ion currents according to a drain voltage V _D and a gate voltage V _G of an ion transistor of a device for DNA analysis according to the inventive concept. This is the result of using a nanopore membrane having the same structure as in the conductance measurement of FIG. 5 and using potassium chloride (KCl) at a concentration of 10 ⁻⁴ M.

Referring to FIG. 6A, the characteristics of the ion current I _D according to the drain voltage V _D when the gate voltage V _G changes from −1 V to 1 V are illustrated, and the characteristics of the diode are shown. This is due to the asymmetrical shape of the fabricated nanopores.

Referring to FIG. 6B, when the drain voltage V _D changes from 0V to 1V, characteristics of the ion current I _D according to the gate voltage V _G are shown. The graph inserted therein is a graph showing the value of the ion current I _D on a log scale. It can be seen that the ion current I _D is controlled according to the gate voltage V _G , and the ion transistor behaves similarly to a conventional p-type field effect transistor (pFET). The ions flowing into the nanopores through the flow of the ion current I _D at the negative gate voltage V _G may be considered to have a positive charge, and may be potassium ions (K +) in a potassium chloride (KCl) solution. Titanium oxide (TiO ₂ ), which is the second insulating layer 24, has a negative surface charge, and thus, K +, which is a cation, may be filled in the nanopores 25 and flow through the channel, and the mobility of ions may be about 1 cm ^2. It was measured at / secV.

Figure 7a is a schematic diagram showing the DNA passing through the nano-pores 25 in accordance with the spirit of the present invention, Figure 7b is a graph showing the flow of current to explain the DNA sequencing method.

Referring to FIG. 7A, the DNA in the solution containing the DNA passes through the nanopores 25 as shown. When the DNA contained in the solution passes through the nanopores 25, there is an energy barrier due to elements such as electrostatic interaction or geometric limitations in the nanopores 25. By the voltage applied to the electrode layer 21 of the solution and the nano-pore membrane 20, DNA can pass in one direction to overcome the barrier. DNA is a polymer of nucleotides, and the nucleotide is composed of pentose sugar, phosphoric acid and base, and the phosphate group has a negative charge due to the difference in electronegativity between phosphorus and oxygen constituting the phosphate group. Therefore, the movement can be adjusted by the ion field effect mentioned above. The charge of the DNA, RNA, peptide or protein to be analyzed can be controlled by using a high charge or a specific charge to have a specific charge. When the DNA passes through the nano-pores (25), the current is blocked by the ions flowing in the nano-pores (25) to generate a blockade signal (blockade signal), whereby the adenine (A), the constituent base of the DNA, According to the type of thymine (T), guanine (G), and cytokine (C), a second electrical signal, for example, a current value, may be displayed. The current may be measured from the first electrode 30 and the second electrode 40 of the DNA analysis apparatus 100 of FIG. 1, through which the base of the DNA may be analyzed.

In general, when using the nano-pores 25 of 10nm or less, it is possible to distinguish between single stranded and double stranded DNA (double stranded) of the DNA, according to the DNA analysis apparatus according to the present invention 10nm or less Since the nano pores 25 are used, such a distinction may be possible. For the analysis of DNA, the transit time through which the DNA passes through the nanopores 25 should be sufficient. In the device for DNA analysis according to the present invention, by controlling the first electrical signal applied, for example, a voltage, by controlling the charge in the nano-pores 25 corresponding to the channel of the ion transistor, it is possible to control the rate of DNA movement have. A detailed control process will be described below with reference to FIG. 8.

Referring to FIG. 7B, an exemplary method of sequencing DNA base sequences using a second electrical signal, such as the current received by the blocking signal, is shown. When the second electrical signal is referred to as a detection current, each of the bases A, T, G, and C constituting the DNA may represent a detection current having a different magnitude. Therefore, the sequential detection of each base of the DNA is possible through the magnitude of the detection current, thereby enabling the sequencing of the base sequence of the DNA. In this case, for easy analysis of the second electrical signal, it is required that the amount of current detected is large and the difference in the current value according to the base sequence is large. In the device for DNA analysis according to the present invention, by controlling the gate voltage (V _G ) and the surface charge of the second insulating layer 24 applied to the conductive layer 21 of the nano-pore membrane 20 of FIG. 7A, The amount of current passing through the nanopores 25 may also be adjustable, thereby facilitating sequencing of DNA.

8 is a flowchart illustrating an exemplary DNA analysis process using a device for DNA analysis using a nano-pore structure according to the spirit of the present invention.

Referring to FIG. 8 together with FIG. 1, after preparing the device for DNA analysis 100 of FIG. 1, accommodating a solution in the chamber 10, a first signal in the chamber 10 in the electrical signal unit 50 may be used. In operation S110, a first electrical signal, for example, a predetermined voltage is applied to the electrode layer 21 of the electrode 30, the second electrode 40, and / or the nanopore layer 20. For example, a positive gate voltage V _G and a drain voltage V _D and a negative source voltage V _S may be applied. As a result, the step S115 of moving the DNA in the solution of the chamber 10 proceeds, and the DNA may be moved from the first region 12 of the chamber 10 to the second region 14. The gate voltage V _G may be 1 V or less so that the first insulating layers 22a and 22b and the second insulating layer 24 of the nanopore film 20 do not breakdown or leak current flows. In the step S110, a separate voltage may not be applied to the gate voltage V _G.

Next, an operation (S120) of receiving and analyzing a second electrical signal, for example, a current, from the electrical signal unit 50 is performed. The second electrical signal may be a current generated by the flow of ions. When the DNA passes through the nano-pores 25, the above-described blocking signal is generated, through the reception of the current, it is possible to detect the DNA and sequencing the base sequence. If detection of DNA is recognized, it may be necessary to slow down or stop the movement for analysis of DNA.

In the next step, step S130 of adjusting the gate voltage V _G , which is the first electrical signal applied to the conductive layer 21 of the nano-pore film 20, is performed. This is to control the movement of DNA for analysis after detecting the DNA through the current received in the receiving and analyzing step (S120). In step S130, the step S135 of controlling the moving speed of the DNA in the nanopores 25 is performed. For example, when a positive voltage is applied to the gate voltage V _G , the movement of DNA having a negative charge may be stopped or the movement speed may be reduced according to the magnitude of the voltage. Therefore, it is possible to secure a transit time for analysis of DNA. In addition, even when DNA is not detected in the reception and analysis step (S120), the entry of DNA into the nanopores 25 may be prevented as necessary. For example, when a negative voltage is applied to the gate voltage V _G , the DNA may be blocked from moving into the nanopores 25.

While controlling the movement of DNA, the analysis of DNA is performed again through the step of receiving and analyzing current (S120), controlling the gate voltage (V _G ) (S130), controlling the movement of DNA (S135), and receiving a current. The process of analyzing (S120) may be repeated.

9 is a schematic diagram showing a PCR quantitative detection apparatus using a nano-pore structure according to an embodiment of the present invention.

Polymerase chain reaction (PCR) is a technique for copying and amplifying specific portions of DNA. Various techniques, such as real-time PCR apparatus, are used for quantitative analysis of amplified DNA.

9, the PCR quantitative detection apparatus 1000 using the nano-pore structure includes a buffer solution input unit 210, a sample input unit 220, a reaction unit 230, a concentration control unit 240, and a detection unit 250. ) And a disposal unit 260. A plurality of opening and closing portions are positioned between the buffer solution input unit 210, the sample input unit 220, the reaction unit 230, the concentration control unit 240, the detection unit 250, and the disposal unit 260, and independently May be performed.

The buffer solution input unit 210 may inject a buffer solution into the reaction unit 230 and the concentration controller 240. The buffer solution may provide a buffer solution or dilution solution for the amplification and detection environment composition of DNA. The buffer solution inlet 210 may be configured in the form of a syringe, in which case it may be controlled and operated by pneumatic pressure, such as a syringe pump. The buffer solution input unit 210 may include a plurality of the syringes.

The sample input unit 220 may input samples for amplification of the DNA into the reaction unit 230. The sample may be a template for amplifying DNA, primers, DNA nucleotides, DNA polymerases, and the like. Sample input unit 220 may also be configured in the form of a syringe.

The reaction unit 230 is a chamber in which PCR is performed and may have a size of several nanoliters to several microliters. The reaction unit 230 may receive a material necessary for performing PCR from the buffer solution input unit 210 and the sample input unit 220 to perform a reaction. The reaction unit 230 may include a peltier element, a resistance heater, or the like, whereby the temperature required to perform PCR may be controlled.

The concentration control unit 240 may control the concentration of the DNA amplified by the reaction unit 230 by the buffer solution from the buffer solution input unit 210 to optimize the detection of the DNA in the detection unit 250. The concentration controller 240 may be configured in the form of a channel as shown in the figure, and may dilute the DNA by a dilution solution such as potassium chloride (KCl) solution.

The detector 250 may include a device for DNA analysis 100 using the nanopore structure of FIG. 1. DNA moved by electrophoresis may be quantitatively analyzed while passing through the nanopores in the detector 250. Similar to the sequencing of the DNA described above with reference to FIGS. 7A and 7B, the detection unit 250 may analyze the length of the DNA and the amount of DNA by the change of the ion current caused by the DNA passing through the nanopores.

According to the PCR quantitative detection apparatus 1000 using the nanopore structure of the present invention, the absolute quantitative analysis is possible by analyzing the DNA through the nanopore. In addition, quantitative analysis may be performed in a short time through dilution after amplification, and may be analyzed at low cost since it is a non-labeled method. In the present embodiment, the case of quantitatively analyzing DNA amplified by PCR, but the present invention is not limited to this, it will be understood that it can be used for quantitative analysis of DNA in various applications.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Will be clear to those who have knowledge of.

10 chamber 12 first region of chamber
14 second region of chamber 20 nanopore membrane
20a: laminated film 21: conductive layer
22a, 22b: 1st insulating layer 24: 2nd insulating layer
25a: Opening 25: Nano Pore
30: first electrode 40: second electrode
50: electrical signal unit 60: substrate
70: first mask layer 80a, 80b: second mask layer
90 photoresist layer

Claims (13)

A chamber containing a solution, the chamber comprising a first region and a second region;
A first electrode positioned in the first region;
A second electrode positioned in the second region opposite the first electrode; And
A nano pore film disposed between the first electrode and the second electrode and including a conductive layer and a nano pore passing through the conductive layer;
And an electrical signal unit electrically connected to the conductive layer, the first electrode, and the second electrode to apply a first electrical signal and receive a second electrical signal therefrom.
DNA analysis device using a nano-pore structure, characterized in that for detecting the DNA in the solution using the second electrical signal. The method according to claim 1,
And detecting one or more bases constituting the DNA using the second electrical signal that changes when the DNA passes through the nanopores. The method of claim 2,
DNA detection device using a nano-pore structure characterized in that to sequentially detect the second electrical signal having a different value according to the base constituting the DNA, the sequencing of the DNA. The method according to claim 1,
The nano-pore membrane further comprises a first insulating layer laminated on the upper, lower or both sides of the conductive layer. 5. The method of claim 4,
The nano-pore film further comprises a second insulating layer formed on the side of the conductive layer adjacent to the nano-pore and the side of the first insulating layer and surrounding the first insulating layer. DNA analysis apparatus using. 6. The method of claim 5,
The conductive layer may include titanium nitride (TiN), the first insulating layer may include silicon oxide (SiO ₂ ), and the second insulating layer may include titanium oxide (TiO ₂ ). DNA analysis device using the structure. The method according to claim 1,
The device for DNA analysis is a device for DNA analysis using a nano-pore structure, characterized in that the electric signal portion operates as an ion field transistor by a voltage applied to the conductive layer, the first electrode and the second electrode. The method according to claim 1,
The nano-pore is a device for DNA analysis using a nano-pore structure, characterized in that the cylindrical shape of the upper and lower sides are the same or the truncated conical shape of the diameter of the upper and lower sides. Preparing a device for DNA analysis of any one of claims 1 to 8;
Receiving a solution containing DNA in the chamber of the DNA analysis device;
Applying a first electrical signal to the conductive layer, the first electrode, and the second electrode from the electrical signal portion; And
Receiving the second electrical signal generated when the DNA passes through the nano-pores; DNA analysis method using a nano-pore structure comprising a. 10. The method of claim 9,
Receiving the second electrical signal,
And changing and applying the first electrical signal applied to the conductive layer, in order to control the movement speed of the DNA. The method of claim 10,
DNA moving method using a nano-pore structure, characterized in that the movement speed of the DNA is reduced as the first electrical signal applied to the conductive layer is increased. At least one input unit providing a sample and a buffer solution containing DNA;
A reaction unit in which amplification of the DNA is performed; And
Claims 1 to 8 of the DNA analysis device according to any one of the claims, wherein the nano amplification comprising a detector for quantitatively analyzing the DNA through the electrical signal generated while passing through the nano-pores PCR quantitative detection apparatus using a pore structure. The method of claim 12,
Located between the reaction unit and the detection unit, receiving the buffer solution from the input unit, PCR quantitative detection device using a nano-pore structure further comprises a concentration control unit for controlling the concentration of the amplified DNA .

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