• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The present invention includes a sample reservoir; A micro processing channel directly connected to the sample storage part and serving as an inflow passage of a sample; And, the nucleic acid analysis apparatus and nucleic acid analysis method comprising the temperature control-detection unit is mounted at the same time one or more MOSFET-type transistor and a heater and a temperature sensor at the same time inside the "micro processing channel". According to the present invention, nucleic acid mutations are detected by detecting denatured double-stranded nucleic acid or renatured double-stranded nucleic acid at the Tm temperature of the nucleic acid attached to the MOSFET transistor. In particular, it is possible to effectively detect a single nucleotide variation (SNP) of the nucleic acid.
    公开号:KR20020090734A
    申请号:KR1020010029729
    申请日:2001-05-29
    公开日:2002-12-05
    发明作者:임근배
    申请人:삼성전자 주식회사；
    IPC主号:
    专利说明:

    Device for assaying mutation of nucleic acids and Method for assaying nucleic acids using the device
    [4] The present invention relates to a nucleic acid analysis apparatus useful for detecting mutations in nucleic acids, in particular single nucleotide variations (SNPs) of nucleic acids, more particularly sample reservoirs; A micro-processing channel directly connected to the sample storage part to be an inflow passage of a sample; And a temperature control-detection unit equipped with at least one 'nucleic acid' fixed MOSFET transistor and a heater and a temperature sensor in the micro-processing channel, and a method for analyzing a mutation of nucleic acid using the same.
    [5] Single nucleotide variation (SNP) of nucleic acid is the single base-pair variation in DNA between individuals and is the most common form of DNA sequence polymorphism (about 1kb). Since SNPs cause a difference in sensitivity to individual diseases, they can be effectively used for diagnosis, treatment and prevention of genetic diseases. Therefore, there has been a need for a quick and easy way to find SNPs, which are parts of genes that bring about individual differences, race differences, and "sensitivity" differences to diseases.
    [6] In order to detect SNPs, the observation of separation of DNA double-stranded structure with temperature is most common. At temperatures above 96 ° C, all the hydrogen bonds in the double stranded DNA are broken and separated. These traits can be used to identify mutated DNA sequences, but using different equipment and machinery, and the technology to measure the separation of DNA in real time by accurate and precise temperature control is very difficult.
    [7] In US Pat. No. 6,171,850 (Integrated devices and systems for performing temperature controlled reactions and analyses), heat control was used to direct temperature control inside and outside the reactor, respectively. The reaction system consists of a heater and one or more heat exchangers. However, the system only mentions the temperature control of several reactors, and includes a heat exchanger, making it difficult to expect DNA detection and channel characteristics.
    [8] U.S. Patent No. 6,174,675 (Electrical current for controlling fluid parameters in micro-channels) is equipped with an electrically controlled heater and a cooler directly in the flow path and shows various types of heaters. However, this patent discloses only a temperature control device used for PCR and the like, and mentions no device for detecting the separation of DNA double strand structure with temperature.
    [9] U.S. Patent No. 5,683,657 (DNA meltometer) discloses a double-stranded DNA separated at a temperature of Tm, a temperature control chamber that maintains a constant temperature and can carry buffer solutions, heating and cooling means for temperature control of the chamber, and Tm. Disclosed is a nucleic acid analysis device composed of means for labeling with a fluorescent material and detecting the same. However, this device has a problem in that the DNA detection means is separate from the temperature control chamber and thus the configuration is complicated and the detection time is delayed.
    [10] Thus, the present inventors constructed a system capable of various temperature control and DNA detection on a small device using a principle that the DNA formed in a double-stranded structure is divided into two strands as the temperature increases, thereby mutating the DNA. The (SNP) measuring device was completed. According to the present invention, it is possible to determine in real time whether the DNA is separated or not through a detection device manufactured directly in the flow path.
    [11] It is therefore an object of the present invention to provide a novel nucleic acid analysis apparatus capable of real time detection of mutant DNA by accurate and precise temperature control by micro-processing a temperature control device and a detection device directly in a flow path.
    [12] It is also an object of the present invention to provide a method that can effectively detect the SNP using the nucleic acid analysis device.
    [1] 1 is a schematic view of a nucleic acid analysis device of the present invention including a temperature control-detection unit on a bottom surface of a microfabrication channel.
    [2] Fig. 2 schematically shows the nucleic acid analysis device of the present invention including a temperature control-detection unit having a MOSFET-type transistor of fixed nucleic acid on a side wall or an edge of a microprocessing channel and having a heater and a temperature sensor on the upper surface of the microprocessing channel. Drawing
    [3] 3 is a view showing a fabrication process of the MOSFET transistor of the present invention.
    [13] In order to achieve the above object, in the present invention, a heater, a temperature sensor, and a DNA detection sensor (MOSFET type) are directly built in a flow path (micro-processing channel) through which a fluid can flow for separation and detection of DNA according to temperature. Produced at the same time with (built-in) According to this, while controlling the temperature on the DNA with a heater and a temperature sensor, it is possible to determine in real time whether the DNA is a double-stranded or single-stranded structure with a DNA detector.
    [14] Specifically, the present invention includes: ① a sample reservoir; ② a micro-processing channel is directly connected to the sample reservoir, the inlet passage of the sample; And a temperature control-detection unit simultaneously equipped with at least one nucleic acid-fixed MOSFET-type transistor, a heater, and a temperature sensor in the microprocessing channel, wherein the heater and the temperature sensor are nucleic acid attached to the transistor. Controlling the temperature at which each of the transistors is denatured double-stranded nucleic acid or renatured double-stranded nucleic acid at the Tm temperature of the nucleic acid attached thereto. It provides a nucleic acid analysis device characterized in that the detection.
    [15] In the present invention, a metal-oxide-semiconductor field-effect transistor (MOSFET) is used as a sensor for detecting charge changes before and after separation of a double-stranded nucleic acid. It was.
    [16] Preferably, the MOSFET transistor of the present invention is characterized in that it is located at the sidewall or convex corner of the microfabricated channel. All biomolecular sensors using FETs manufactured to date have been fabricated on a plane. However, the biomolecular sensor fabricated in the present invention is a three-dimensional MOSFET form using bulk micromachining and diffusion, and is mainly located at the convex corner of the micromachining channel. The biomolecular sensor of this structure is located directly in the flow path where the nucleic acid flows, which not only significantly shortens the detection time but also greatly reduces the space occupied by the detector, so that more sensors can be installed in a small space. (Reference: Korea Patent Application No. 201-21752, filed by this applicant).
    [17] In the present invention, the MOSFET type transistor has a gold thin film formed in the source and drain regions, and a thiol group immobilized on the gold surface by a self-assembled monolayers (SAM) method. It is characterized by having a nucleic acid of. In the present invention, the sensor has two terminals, a source and a drain, and a thin oxide film on the surfaces of the two terminals, and when Au is attached thereto, a structure of MOS (metal-oxide-semiconductor) is formed. In addition, thiol is attached to the ends of the nucleic acid molecules so that the nucleic acid molecules can be adsorbed with high selectivity between Au and the oxide film. Thiol-attached nucleic acid molecules can be adsorbed onto gold surfaces by SAM (Self-Assembled Monolayers) method. SAM stands for regularly ordered organic monomolecular membranes spontaneously coated on the substrate surface and does not require any external fabrication equipment because of the direct chemical bonds between the substrate and organic molecules. Bio-molecule sensors manufactured to date have difficulty in attaching biomolecules only to specific regions and have weak adhesion. However, the selective adsorption method of thiol-substituted biomolecules on gold using the SAM method used in the present invention not only solves these problems, but also directly changes the amount of charge before and after the reaction because it is directly attached to the surface of the sensor. It is also excellent in finding out.
    [18] In the present invention, the nucleic acid (nucleic acid) immobilized on the MOSFET-type transistor is characterized in that the single stranded (single stranded) DNA, single stranded RNA or single stranded PNA. These single stranded nucleic acids serve as probes to hybridize with the target nucleic acid injected into the sample reservoir. It is preferable that nucleic acids of different base sequences are attached to the transistor.
    [19] In the present invention, the heater functions to increase the temperature of the temperature control-detecting unit, and the temperature sensor functions to control the operation of the heater. Such heaters and temperature sensors are preferably located in close proximity to the transistor in order to effectively control the temperature affecting the nucleic acid attached to the transistor.
    [20] In order to achieve another object, the present invention comprises the steps of: immobilizing a single-stranded nucleic acid probe on the surface metal of the MOSFET-type transistor; (2) injecting a target nucleic acid reacting with the immobilized probe into a sample reservoir, and moving the target nucleic acid along the flow path of the microprocessing channel to the MOSFET transistor; ③ hybridizing a target nucleic acid with a nucleic acid probe of the transistor; ④ gradually raising the temperature to separate the double stranded (ds) nucleic acid into single stranded (ss); And (5) measuring the electric field applied to the gate of the MOSFET transistor.
    [21] It is a method of detecting mutations while raising the temperature after hybridization of nucleic acid at low temperature.The temperature control device and the detection device manufactured in the microfabricated flow path are used to detect the mutation of nucleic acid by precise and precise temperature control. Analyze in real time.
    [22] Alternatively, the present invention comprises the steps of: immobilizing a single stranded nucleic acid probe on a surface metal of a MOSFET-type transistor; (2) injecting a target nucleic acid reacting with the immobilized probe into a sample reservoir, and moving the target nucleic acid along the flow path of the microprocessing channel to the MOSFET transistor; ③ maintaining the target nucleic acid and the nucleic acid probe at a temperature effective to prevent hybridization; ④ gradually lowering the temperature to make single stranded (ss) nucleic acid double stranded (ds); And (5) measuring an electric field applied to a gate of the MOSFET transistor.
    [23] This is a method of detecting mutations by lowering the temperature after denaturing the nucleic acid at a high temperature.
    [24] The apparatus and method of the present invention can be effectively used to detect nucleic acid mutations, in particular single nucleotide mutations (SNPs) of nucleic acids. DNA is double-stranded, which splits into two strands when the temperature is above 96 degrees. The two strands each denature at a relatively high temperature if they perfectly match the base pairs, and denature at relatively low temperatures if there are mismatches. By combining MEMS technology with this principle, we make a system that consists of a very small temperature control device and a detection device that can be controlled at different temperatures. Using such a system, each MOSFET type transistor can be arranged differently by one by one to find out how many degrees of denaturation has occurred at each temperature control device, and can be used to determine the single point mutation of nucleic acid. mutations), and the like.
    [25] Fig. 1 is a diagram schematically showing a case where the temperature control-detecting unit is mounted on the bottom of the micro-processing channel as an example of the nucleic acid analyzing apparatus of the present invention. (a) shows a matched state and (b), (c) and (d) mismatched states. Fig. 2 shows another example of the nucleic acid analysis device of the present invention, in which a MOSFET-type transistor (Electrode, Gold Gate) constituting a temperature control-detecting unit is mounted on a side wall or an edge in a micro processing channel, and a heater and a temperature sensor are shown. (Micro Tmperature Sensor) is a diagram schematically showing the case where it is mounted on the upper surface of the micro processing channel.
    [26] Hereinafter, a method for manufacturing a nucleic acid analysis apparatus of the present invention will be described in detail with reference to the drawings.
    [27] 1. Preparation of micro processing channel
    [28] The body of the device of the present invention may be assembled using methods and materials that are generally suitable for microfabrication techniques. For example, the body of the device of the present invention may comprise a plurality of planar membranes of injection molded portions of various polymer materials or crystalline substrates such as silicon, glass, and the like. In the case of crystalline substrates such as silica, glass or silicon, etching, milling, drilling and the like can be used to create wells and various channels in the device. Microassembly techniques used in the semiconductor industry can be applied to these materials or methods. These techniques include, for example, electrodeposition, low-pressure vapor deposition, photolithography, etching, laser drilling and the like.
    [29] Etching of the substrate using ore plate printing is particularly suitable for the microassembly of the present invention. For example, the first substrate is overlaid with a photoresist, irradiated with electromagnetic light through a lithography mask to expose the photoresist in a pattern such as chambers and / or channels of the device, and the exposed substrate is etched to provide the desired wells and channels. After that, another planar film is covered over the first substrate and bonded. Generally preferred photoresists include polymethyl methacrylate (PMMA) and derivatives thereof, electron beam resists such as poly (olefin sulfone), and the like.
    [30] Preferably, the body of the present invention may be made by combining a portion of an injection molded plastic or the like and a portion of an etched silica or silicon planar film. For example, the sample reservoir of the present invention is formed by injection molding technology, while the micromachining channel and temperature control detector can be microassembled by etching on flat glass, silica or silicon chips or substrates.
    [31] 2. Manufacturing of temperature control unit
    [32] In the present invention, a heater and a temperature sensor are built in the microprocessing channel to control the temperature affecting the nucleic acid attached to the DNA detection unit (MOSFET transistor).
    [33] As the heater, a thin film resistive heater or the like, which is known in the art, may be used. This heater can be manufactured by applying a metal film connected to a power source to the bottom, top or inside of the micromachining channel. Temperature sensors also include thermocouples with bimetal junctions that produce temperature-dependent electric motor forces (EMFs), resistive thermometers including materials with a temperature proportional electrical resistance, thermistors, IC temperature sensors, and quartz ( quartz) and the like can be used.
    [34] 3. Preparation of DNA Detection Part
    [35] Fig. 3 shows the fabrication process of the DNA detection sensor (MOSFET transistor) fabricated in the present invention. First, the nucleic acid is moved to the detection site of the sensor by a micro channel connected to the reservoir. The sensor of the detector is wet etched using an n-type silicon substrate, and then diffuses boron into the etched sidewall to form a p-type silicon region. When the boron region is cut again by wet silicon etching, a source and a drain are formed. To form a MOSFET structure, a thin dry oxide film is grown in the source and drain regions, and chromium is deposited on the oxide film to increase the adhesion property of the gold oxide film, and then gold is deposited thereon. To fix the nucleic acid probes on the surface of the sensor, substitute a thiol group at the end of the nucleic acid. Nucleotides with thiol groups selectively bind only to the gold region present on the sensor surface by SAM method. Therefore, a detection sensor can be manufactured by forming a gold thin film on a MOSFET-type DNA sensor and attaching nucleic acid thereon. In particular, the thiol SAM film attached on the gold thin film can be easily made and reproducible, and can have the maximum applicability by varying thiol's other reactor. In addition, relatively thinner and more aligned thin films can be obtained than other SAM thin films, and because of their strong bonding strength, they are widely used because they are stable to various reactions after forming SAM thin films.
    [36] As described above, according to the present invention, by directly building a heater, a temperature sensor, and a DNA detection sensor in a built-in simultaneously in a flow path through which a fluid can flow, the denaturation phenomenon of nucleic acid according to temperature is generated in real time. It is possible to discriminate and to effectively detect mutations of many kinds of nucleic acids, especially single nucleotide variations (SNPs) of nucleic acids.
    [37] In other words, in order to uncover the mutated DNA sequences, it was necessary to use different equipment and machines for separating and observing the DNA double-strand structure according to the temperature, and to separate the DNA by accurate and precise temperature control in real time. Although it was very difficult to measure, according to the present invention, the detection of mutant DNA by accurate and precise temperature control was performed in real time by simply injecting DNA into the microfabricated flow path and the temperature control device fabricated in the microfabricated flow path. You can do In addition, the temperature distribution appears more precisely in the micro-domain, which results in much more accurate characteristics.
    权利要求:
    Claims (7)
    [1" claim-type="Currently amended] ① sample reservoir;
    (2) a microprocessing channel directly connected to the sample storage unit and serving as an inflow passage of a sample; And,
    (3) includes a temperature control-detection unit simultaneously equipped with at least one nucleic acid-fixed MOSFET transistor, a heater, and a temperature sensor in the microprocessing channel;
    The heater and temperature sensor control the temperature influencing the nucleic acid attached to the transistor, each transistor denatured or restored double-stranded nucleic acid at the Tm temperature of the nucleic acid attached thereon. Nucleic acid mutation analysis device, characterized in that for detecting.
    [2" claim-type="Currently amended] The apparatus of claim 1, wherein the MOSFET transistor is located at a sidewall or a corner of a microprocessing channel, and the heater and the temperature sensor are located in close proximity to the transistor.
    [3" claim-type="Currently amended] The method of claim 1, wherein the MOSFET transistor is a gold thin film formed in the source (drain) source and drain (drain) region, the nucleic acid having a thiol group attached to the surface of the gold immobilized by self-assembled monolayers (SAM) method Nucleic acid mutation analysis device, characterized in that it has a.
    [4" claim-type="Currently amended] The nucleic acid mutagenesis analysis apparatus according to claim 1, wherein the nucleic acid immobilized on the MOSFET-type transistor is single-stranded DNA, single-stranded RNA or single-stranded PNA.
    [5" claim-type="Currently amended] (1) immobilizing single-stranded nucleic acid probes on the surface metal of the MOSFET transistor;
    (2) injecting target nucleic acid reacting with the immobilized probe into the sample storage unit and moving the target nucleic acid along the flow path of the microprocessing channel to the MOSFET transistor;
    ③ hybridizing a target nucleic acid with a nucleic acid probe of the transistor;
    ④ gradually raising the temperature to separate the double stranded (ds) nucleic acid into single stranded (ss); And,
    5. A method for analyzing a mutation of a nucleic acid, comprising measuring an electric field applied to a gate of a MOSFET transistor.
    [6" claim-type="Currently amended] (1) immobilizing single-stranded nucleic acid probes on the surface metal of the MOSFET transistor;
    (2) injecting target nucleic acid reacting with the immobilized probe into the sample storage unit and moving the target nucleic acid along the flow path of the microprocessing channel to the MOSFET transistor;
    ③ maintaining the target nucleic acid and the nucleic acid probe at a temperature effective to prevent hybridization;
    ④ gradually lowering the temperature to make single stranded (ss) nucleic acid double stranded (ds); And,
    5. A method for analyzing nucleic acid mutations comprising measuring an electric field applied to a gate of a MOSFET transistor.
    [7" claim-type="Currently amended] 7. The method according to claim 5 or 6, wherein a single base mutation (SNP) of the nucleic acid is detected.
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    同族专利:
    公开号 | 公开日
    KR100438822B1|2004-07-05|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2001-05-29|Application filed by 삼성전자 주식회사
    2001-05-29|Priority to KR20010029729A
    2002-04-23|Priority claimed from JP2002583675A
    2002-12-05|Publication of KR20020090734A
    2004-07-05|Application granted
    2004-07-05|Publication of KR100438822B1
    优先权:
    申请号 | 申请日 | 专利标题
    KR20010029729A|KR100438822B1|2001-05-29|2001-05-29|Device for assaying mutation of nucleic acids and Method for assaying nucleic acids using the device|
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