WO2011162582A2 - Dispositif d'analyse d'adn utilisant une structure nanoporeuse, procédé d'analyse et dispositif de détection quantitative par pcr - Google Patents

Dispositif d'analyse d'adn utilisant une structure nanoporeuse, procédé d'analyse et dispositif de détection quantitative par pcr Download PDF

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WO2011162582A2
WO2011162582A2 PCT/KR2011/004653 KR2011004653W WO2011162582A2 WO 2011162582 A2 WO2011162582 A2 WO 2011162582A2 KR 2011004653 W KR2011004653 W KR 2011004653W WO 2011162582 A2 WO2011162582 A2 WO 2011162582A2
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dna
nano
electrode
electrical signal
conductive layer
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PCT/KR2011/004653
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English (en)
Korean (ko)
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WO2011162582A3 (fr
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김기범
김현미
이민현
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서울대학교 산학협력단
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Priority claimed from KR1020110061325A external-priority patent/KR101267789B1/ko
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Publication of WO2011162582A2 publication Critical patent/WO2011162582A2/fr
Publication of WO2011162582A3 publication Critical patent/WO2011162582A3/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • the DNA may be sequentially sequenced by detecting the second electrical signal having a different value depending on the base constituting the DNA.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • the moving speed of the DNA may be reduced.
  • 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. .
  • 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.
  • 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.
  • 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.
  • FIG. 1 is a perspective view showing a device for DNA analysis using a nano-pore structure according to an embodiment of the present invention.
  • FIG. 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.
  • FIGS. 4A and 4B are photographs showing a state in which nanopores are formed according to the present invention.
  • FIG. 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
  • FIG. 7B is a graph showing the flow of electric current to explain the method of sequencing DNA.
  • FIG. 8 is a flowchart illustrating a DNA analysis process using a device for DNA analysis using the nanopore structure according to the present invention.
  • FIG. 9 is a schematic diagram showing a PCR quantitative detection apparatus using a nano-pore structure according to an embodiment of the present invention.
  • first, second, etc. are used herein to describe various members, parts, regions, layers, and / or parts, these members, parts, regions, layers, and / or parts are defined by these terms. It is obvious that not. These terms are only used to distinguish one member, part, region, layer or portion from another region, layer or portion. Thus, the first member, part, region, layer or portion, which will be discussed below, may refer to the second member, component, region, layer or portion without departing from the teachings of the present invention.
  • FIG. 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.
  • 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) has almost no difference in ion mobility between cations and anions.
  • the apparatus may further include an injection unit (not shown) and a discharge unit (not shown) capable of injecting and discharging the solution in the chamber 10 from the outside of the chamber 10.
  • the chamber 10 may have a small capacity, and the length of either direction may have a dimension 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.
  • the first electrode 30 and the second electrode 40 may each be a single layer or a composite layer.
  • 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.
  • 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.
  • 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.
  • the portion connected to the conductive layer 21 may be connected by a probe (not shown).
  • the device for DNA analysis 100 is an ionic field effect transistor (IFET).
  • IFET ionic field effect transistor
  • 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.
  • 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.
  • FIG. 2 is a perspective view showing the structure of the nano-pore membrane 20 according to the present invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • RIE reactive ion etching
  • 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.
  • KOH potassium hydroxide
  • 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.
  • PMMA poly methyl methacrylate
  • a two-step etching process of forming the opening 25a in the center of the stacked film 20a is performed.
  • the first mask layer 70 is etched using the second mask layer 80a on which the pattern is formed.
  • the etching may use RIE.
  • the laminated film 20a is etched using the patterned first mask layer 70.
  • 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.
  • the second insulating layer 24 may be deposited to form the nanopore film 20 of FIG. 2.
  • Atomic layer deposition may be used as the deposition method of the second insulating layer 24.
  • ALD Atomic layer deposition
  • CVD chemical vapor deposition
  • 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).
  • 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.
  • 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.
  • FIGS. 4A and 4B are photographs 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.
  • FIG. 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.
  • KCl potassium chloride
  • 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.
  • TiN titanium nitride
  • TiO 2 titanium oxide
  • 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.
  • Debye leghth which is inversely proportional to the square root of the concentration of the electrolyte in the solution.
  • 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 in which the saturation of the ion conductance occurs. This ion selectivity is the basis for operating as an ion transistor.
  • the electrical double layer 26 may overlap in the nano-pores 25 even if the draw length is small.
  • 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.
  • KCl potassium chloride
  • 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.
  • 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.
  • 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.
  • the DNA in the solution containing the DNA passes through the nanopores 25 as shown.
  • 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.
  • 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.
  • 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.
  • 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.
  • the transit time through which the DNA passes through the nanopores 25 should be sufficient.
  • the first electrical signal applied for example, a voltage
  • 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.
  • 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.
  • 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.
  • 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 may also be adjustable, thereby facilitating sequencing of DNA.
  • FIG. 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.
  • a first signal in the chamber 10 in the electrical signal unit 50 may be used.
  • 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.
  • a positive gate voltage V G and a drain voltage V D and a negative source voltage V S may be applied.
  • 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.
  • the second electrical signal may be a current generated by the flow of ions.
  • 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.
  • 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).
  • 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.
  • the entry of DNA into the nanopores 25 may be prevented as necessary.
  • the DNA may be blocked from moving into the nanopores 25.
  • 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.
  • FIG. 9 is a schematic diagram showing a PCR quantitative detection apparatus using a nano-pore structure according to an embodiment of the present invention.
  • PCR Polymerase chain reaction
  • 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.
  • KCl potassium chloride
  • 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.
  • the absolute quantitative analysis is possible by analyzing the DNA through the nanopore.
  • 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.
  • 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.

Abstract

L'invention concerne un dispositif d'analyse d'ADN utilisant une structure nanoporeuse, un procédé d'analyse d'ADN et un dispositif de détection quantitative par PCR. Selon une forme de réalisation de l'invention, le dispositif d'analyse d'ADN utilisant une structure nanoporeuse comprend: une chambre recevant une solution et qui comporte une première zone et une seconde zone; une première électrode positionnée dans la première zone; une seconde électrode positionnée dans la seconde zone, face à la première électrode; un film nanoporeux, positionné entre la première électrode et la seconde électrode et qui comporte une couche conductrice et des nanopores pénétrant dans la couche conductrice; et une partie signaux électriques connectée à la couche conductrice, à la première électrode et à la seconde électrode et qui applique à ceux-ci des premiers signaux électriques et reçoit de ceux-ci des seconds signaux électriques, l'ADN présent dans la solution étant détecté au moyen des seconds signaux électriques.
PCT/KR2011/004653 2010-06-25 2011-06-27 Dispositif d'analyse d'adn utilisant une structure nanoporeuse, procédé d'analyse et dispositif de détection quantitative par pcr WO2011162582A2 (fr)

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US9649232B2 (en) 2011-06-10 2017-05-16 The Procter & Gamble Company Disposable diaper having reduced absorbent core to backsheet gluing
WO2018117726A1 (fr) * 2016-12-22 2018-06-28 서울대학교 산학협력단 Appareil et procédé d'extraction d'acide nucléique utilisant un nanofiltre
EP3684724A4 (fr) * 2017-09-22 2021-12-08 Applied Materials, Inc. Formation de pores dans un substrat
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US11691872B2 (en) 2017-09-22 2023-07-04 Applied Materials, Inc. Pore formation in a substrate
WO2021235758A1 (fr) * 2020-05-18 2021-11-25 서울대학교산학협력단 Dispositif et procédé d'extraction de biomolécules utilisant un nanofiltre
WO2022005780A1 (fr) * 2020-07-02 2022-01-06 Illumina, Inc. Dispositifs à transistors à effet de champ
US11898983B2 (en) 2020-07-02 2024-02-13 Illumina, Inc. Devices with field effect transistors
KR20220135619A (ko) * 2021-03-31 2022-10-07 고려대학교 산학협력단 마이크로 포어를 이용한 단일 세포 분석 장치
KR102514030B1 (ko) 2021-03-31 2023-03-24 고려대학교 산학협력단 마이크로 포어를 이용한 단일 세포 분석 장치
CN113176317A (zh) * 2021-04-28 2021-07-27 苏州罗岛纳米科技有限公司 一种单层膜双纳米孔dna检测设备及检测方法
CN113176317B (zh) * 2021-04-28 2024-01-02 苏州罗岛纳米科技有限公司 一种单层膜双纳米孔dna检测设备及检测方法

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