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WO2016126141A1 - Appareil à disque d'extraction d'adn et procédé d'extraction d'adn mettant en œuvre ce dernier - Google Patents

Appareil à disque d'extraction d'adn et procédé d'extraction d'adn mettant en œuvre ce dernier Download PDF

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Publication number
WO2016126141A1
WO2016126141A1 PCT/KR2016/001318 KR2016001318W WO2016126141A1 WO 2016126141 A1 WO2016126141 A1 WO 2016126141A1 KR 2016001318 W KR2016001318 W KR 2016001318W WO 2016126141 A1 WO2016126141 A1 WO 2016126141A1
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WO
WIPO (PCT)
Prior art keywords
chamber
dna
dna extraction
disk
temperature sensitive
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2016/001318
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English (en)
Korean (ko)
Inventor
유재천
임다예슬
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Math Power Co Ltd
Sungkyunkwan University
Original Assignee
Math Power Co Ltd
Sungkyunkwan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Math Power Co Ltd, Sungkyunkwan University filed Critical Math Power Co Ltd
Publication of WO2016126141A1 publication Critical patent/WO2016126141A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to a DNA extraction disk device using a temperature sensitive material and a DNA extraction method using the same. More specifically, the present invention is to break down the cells (cells) and enzymes to extract DNA required for various nucleic acid analysis.
  • the present invention relates to a DNA extraction disk apparatus for heating and controlling a sample at an active temperature of and a DNA extraction method using the same.
  • chambers necessary for various functions are integrated together on the Disc. Only the chamber part responsible for cell destruction function during cell destruction must be selectively temperature controlled. Otherwise, during heating for cell destruction, sites unrelated to cell destruction may be heated or affected simultaneously, causing problems in overall performance.
  • the present invention provides a cell destruction chamber for storing an enzyme and at the same time storing an enzyme to help the cell destruction of the sample;
  • Feedback Provides a DNA extraction disk device that can separate the DNA contained in the cell by controlling and a DNA extraction method using the same.
  • an object of the present invention is to place a temperature sensitive material in the cell destruction chamber to directly measure the temperature of the sample in the cell destruction chamber and thereby to feed back the heating device.
  • the cell extraction process is performed chemically to provide a DNA extraction disk device for separating the DNA in the cell and a DNA extraction method using the same.
  • Another object of the present invention further comprising a silica membrane (silica membrane) for binding to the DNA in the disk to remove the debris (debri) contained in the cells destroyed in the cell destruction chamber by centrifugal force by the disk rotation
  • a silica membrane sica membrane
  • the present invention provides a DNA extraction disk device including a cell destruction step of destroying bacterial cells contained in a sample by a chemical method, a washing step of removing debris, and a DNA extraction step, and a DNA extraction method thereof.
  • DNA is a reverse transcription from deoxyribonucleic acid, RNA, which is obtained from bacteria, viruses, plants, animal body parts (hair, flesh, etc.) or secretions thereof (acupuncture, urine, etc.), blood or food samples. It is characterized in that it is any one selected from cDNA (Complementary DNA) samples obtained by reverse transcription)
  • Bacteria in the sample were taken from food according to Korean Food Standards Codex or FDA food code of the United States, and injected into 225 ml of liquid medium suitable for target pathogens for homogenization. 18-24 hours after enrichment culture, using a colony of separated cultures in a selective agar medium suitable for the target pathogen;
  • Centrifugation to porous filters using pellets of bacteria obtained by centrifugation after carrying out enrichment culture for the target pathogen, or homogenized by stomacher from food. preferably through a filter).
  • DNA extraction disk is used interchangeably with “disk”.
  • DNA extraction discs A drive controller for rotating and driving the DNA extraction disk; A heating device for adjusting the temperature of the cell disruption chamber of the DNA extraction disk; And an optical sensor for measuring the temperature of the cell destruction chamber,
  • the DNA extraction disk is a sample injection port for injecting a sample; And an enzyme for chemically destroying the cells contained in the sample while storing the injected sample, a heating film for absorbing the heat generated from the heating device or transferring the absorbed heat, and a temperature. And a cell disruption chamber consisting of a temperature sensitive chamber that stores a temperature sensitive material.
  • the temperature sensitive material is preferably a paper coated or dyed with a phase change material, a low-melting alloy, or a temperature sensitive fluorescence material.
  • the paper uses a porous membrane or art paper and art paper is preferred in the present invention.
  • the phase change material may be a paraffin-based, non-paraffin-based, inorganic-based divorce-based, metal-based, etc. among the materials that change from the solid phase to the liquid phase.
  • the low temperature melting alloy may be a kind of bismuth (Bismuth) alloy and has a characteristic that the melting temperature varies depending on the blending ratio.
  • Representative low-temperature melting alloys include Wood's metal, Field's metal, Cerrolow 136 and Cerrolow 117.
  • the composition of wood metal is bismuth 50%, lead 26.7%, indium 8.5% and cadmium 10%. It is melted at 70 °C.
  • the composition of the field metal is composed of bismuth 32.5%, tin 16.5%, indium 51%, and melts at 62 °C.
  • Cerrolow 136 alloy is composed of bismuth 29%, lead 18%, tin 12%, indium 21%, and melts at 58 °C.
  • the Cerrolow 117 alloy is composed of bismuth 44.7%, lead 22.6%, tin 8.3%, indium 19.1%, cadmium 5.3% and melts at 47.2 ° C. Fields metal is preferred in the present invention.
  • the temperature sensitive material is preferably arranged in an array in the cell disruption chamber.
  • the heating device is preferably a laser heating device or an induction heating device.
  • the heating film of the present invention is preferably a metal plate, it is further preferred to be heated by the laser heating device further comprises a black coating surface by black paint.
  • the black paint absorbs heat from the laser heater so that heat is transferred through the cell destruction chamber by the metal plate to heat the sample in the cell destruction chamber.
  • the black paint is preferably a heat resistant paint which is not melted by the laser heater.
  • the metal plate is preferably in the range of 0.01 to 0.1 mm aluminum (alumnium) to magnetic metal material.
  • the induction heating device generates a magnetic force line in the coil when an alternating current is sent to the coil, and the magnetic force line causes a eddy current to the metal plate placed on the coil by Faraday's law of electromagnetic induction. It generates and heats a metal plate.
  • the heating film is preferably a black film
  • the black film absorbs laser light from the laser heater to heat the sample in the DNA destruction chamber.
  • the black film is preferably a heat resistant film which is not melted by a laser heating device.
  • the heat resistant film is preferably an aramid film, a polyethylene terephthalate (PET) film, a polyimide film.
  • the heating film is a black coating layer formed on one surface of the cell destruction chamber by black paint.
  • Still another object of the present invention is a silica membrane for binding to DNA from the cells destroyed in the cell destruction chamber;
  • a washing chamber storing a washing solution for washing the silica membrane;
  • a waste chamber for storing debris from the cells destroyed in the cell destruction chamber or for storing impurities from the washing of the silica membrane;
  • An extraction buffer chamber configured to wash DNA bound to the silica membrane by the washing solution, and to store an extraction buffer for extracting DNA bound onto the silica membrane;
  • a DNA chamber for collecting DNA extracted from the silica membrane or for amplifying the collected DNA;
  • a channel for connecting the chambers to provide a passage through which the fluid can flow, and a valve for controlling the flow of the fluid.
  • the fluid is moved along the flow path by the centrifugal force due to the disk rotation.
  • the silica membrane is washed using the washing solution to remove impurities from the silica membrane, and finally, the extraction buffer is introduced into the silica membrane to obtain pure purified DNA when the extraction process is performed. Lose.
  • the purified DNA is moved to the DNA chamber to perform a DNA amplification process as needed.
  • the sample is preferably mixed with a buffer for enhancing the cell destruction mechanism and the silica membrane adsorption force of DNA, and injected into the cell destruction chamber.
  • the buffer aids cell destruction by combining EDTA, glucose, lysozyme, SDS, and the like, which are used for cell destruction, and contains guanidine hydrochloride, Tris-HCL, and the like to bind DNA on the silica membrane.
  • the buffer may be divided into a buffer for cell destruction and a buffer for DNA silica membrane adsorption.
  • the former is called a lysis buffer and the latter is called a binding buffer, but when the two functions are combined, it is usually called a binding buffer, and if necessary depending on the characteristics of the binding buffer.
  • Isopropanol can be added to proceed with bacterial destruction. Isopropanol is responsible for separating cellular proteins and DNA.
  • isopropanol is preferably mixed with a binding buffer and injected into a cell destruction chamber to proceed with cell destruction.
  • the enzyme RnaseA can be added to the sample.
  • Enzyme RnaseA induces the hydrolysis of RNA and removes false positive factors caused by RNA when analyzing DNA extraction results, helping to confirm pure DNA extraction results.
  • the silica membrane may be used in place of a positively charged material, such as a porous plastic or bead having a positive charge.
  • DNA is negatively charged due to the 5'-terminal phosphate group as a whole.
  • the DNA Due to the potential difference between the positively charged material and the negative charge of the DNA, the DNA is attracted to each other and the DNA is adsorbed onto the surface of the positively charged material.
  • the larger the potential difference the greater the electrostatic interaction, resulting in a larger adsorption efficiency.
  • the surface of the positively charged material is preferably introduced to increase the DNA adsorption efficiency by introducing an amine group having a large positive charge value.
  • Another object of the present invention is characterized in that the DNA chamber of the DNA extraction disk stores the enzyme and buffer solution required for the DNA amplification process and performs DNA amplification.
  • the DNA chamber stores a buffer solution containing various enzymes such as primers, including dNTP, and a separate polymer for storage of polymerase. It is preferred to have a laase chamber.
  • the DNA amplification process is performed by repeatedly performing a thermo cycle of a polymer chain reaction (PCR),
  • the PCR is denaturation ( ⁇ 95 o C), annealing ( ⁇ 50 o C), and extension ( ⁇ 72) o This is achieved by running a thermal cycle in the DNA chamber that periodically repeats the three temperatures consisting of C), but the isothermal amplification is performed for about 90 minutes at one specific temperature (eg 60 o C). By heating.
  • the DNA extraction disk device is characterized in that it further comprises a stirrer (stirrer) for mixing the sample of the cell disruption chamber.
  • a stirrer stirrer
  • the stirrer includes a stirring magnet in the cell disruption chamber for sample homogenization and circulation in the cell disruption chamber; And a stirring motor having a small permanent magnet attached to the stirring magnet to perform the stirring operation by exerting an attractive force and a reaction force.
  • the stirrer stores enzymes that assist cell destruction, such as proteinaseK, in a dry powder form or in a buffer solution in a cell destruction chamber, injects a sample into the cell destruction chamber and mixes and homogenizes the enzyme well during the cell destruction process. Be sure to
  • the DNA extraction disk device is characterized in that it further comprises a fluorescent sensor or turbidity sensor (turbidity meter) for quantitatively analyzing the DNA amplification product (product).
  • a fluorescent sensor or turbidity sensor for quantitatively analyzing the DNA amplification product (product).
  • the driving control unit includes a turn table for placing the DNA extraction disk; And a motor for rotating the DNA extraction disk on the turn table.
  • the disk diameter of the disk is preferably 120 mm, 80 mm, 60 mm or 32 mm, and the disc diameter is preferably 1.2 mm to 10 mm.
  • the optical sensor is a photodiode, a camera, a photodiode array, a spectrometer, a charge-coupled device (CCD), a complementary metal-oxide-semiconductor (CMOS) image sensor, a laser It is preferred that any one of the laser power meters is used.
  • the optical sensor may measure the temperature of the sample by measuring the amount of fluorescence from a temperature sensitive fluorescence dye installed in the cell destruction chamber or by measuring the fluidity or transparency of the temperature sensitive material installed in the cell destruction chamber.
  • the optical sensor is also preferably used as a fluorescence sensor or turbidity meter for quantitatively analyzing the DNA amplification product.
  • the temperature-sensitive fluorescent dye may measure the temperature of the sample by measuring the emission intensity of fluorescence according to the excitation of the light emitting diode with the optical sensor. Can be measured.
  • the amount of DNA can be quantitatively measured by measuring the emission intensity of fluorescence according to excitation of the light emitting diode with the optical sensor.
  • the amount of DNA can be quantitatively measured by measuring the turbidity according to excitation of the light emitting diode with the optical sensor.
  • the disk is made of a silicon wafer, polypropylene, polyacrylate, polyvinyl alcohol, polyethylene, polymethyl methacrylate (PMMA), cyclic olefin polymer (COC) and polycarbonate It may be formed of one or more selected from the group. However, plastics are preferred for economic reasons and for ease of processing.
  • the disk is preferably composed of an upper substrate, an intermediate substrate and a lower substrate, which are preferably bonded by an adhesive.
  • the disk is formed by laminating and bonding the upper substrate, the intermediate substrate and the lower substrate, and includes a double-sided adhesive tape for bonding each substrate.
  • the double-sided adhesive tape is surface treated with a special adhesive (gluing agent) on both sides of the release paper such as paper, vinyl, polyester film, polyethylene film, and other synthetic materials, and high sealing according to the conditions required It is possible to select and use a pressure-sensitive adhesive material which has characteristics such as sealing), buffering, vibration damping, impact resistance, heat resistance, adsorption, and adhesion.
  • the double-sided adhesive tape does not use a release paper or backing, and that an adhesive (gluing agent) itself forms the double-sided adhesive tape.
  • the double-sided adhesive tape is preferably in the form of an adhesive (gluing agent) without double-coating the adhesive on a release paper or using a release paper, the adhesive is hot melt (silicone, silicone, rubber, modified silicone, acrylic) , Materials such as polyamide, polyamide, polyolefin, teflon, polyester, epoxy, UV curable adhesive, UV adhesive, thermoplastic resin, and the like may be used.
  • Still another object of the present invention is a DNA extraction disk apparatus according to the present invention, comprising: injecting a sample into a cell destruction chamber through a sample inlet; Heating the cell destruction chamber by a heating device; A sample stirring step of rocking a stirring magnet during the heating step; A temperature measuring step of measuring the temperature of the cell destruction chamber by reading a change in temperature sensitive material (transparency, flowability, fluorescence amount or volume change) through an optical sensor; Controlling the on-off of the heating device according to the temperature value obtained in the temperature measuring step to maintain the activation temperature of the enzyme; Binding to the silica membrane while the DNA destroyed by the heating step passes through the silica membrane under centrifugal force due to disk rotation; A washing step of washing the DNA-bound silica membrane using a washing solution to send impurities to a waste chamber; And after the washing step, the DNA is separated from the silica membrane using an extraction buffer to extract DNA bound to the silica membrane, and then the extracted DNA is sent to a DNA chamber.
  • the DNA extraction method using the DNA extraction disk device is a DNA amplification step for increasing the number of DNA obtained in the extraction step; And a product analysis step of identifying an amplification product by turbidity and fluorescence measurement.
  • the DNA extraction method using the DNA extraction disk device is characterized in that it further comprises a disk authentication step of verifying whether the disc is genuine or reused over the Internet by embedding the RF IC on the disk.
  • the disc of the present invention should not be reused for single use.
  • the ID information of the disk is sent to the server and stored in each step of the disk authentication, so that the disk having the same ID information has been used in the past by checking the history information on the server when the disk is used, thereby reusing the disk. You can automatically check whether or not.
  • the DNA extraction method using the DNA extraction disk device is characterized in that it further comprises a result output step of displaying the result obtained in the output analysis step on the display unit, or providing to the outside through the Internet network. .
  • the DNA extraction method using the DNA extraction disk device after the results sending step, receiving the hygiene score from the public office (for example, the Ministry of Health, Welfare, Education, Food and Drug Administration) through the Internet network remotely billboard It characterized in that it further comprises a hygiene score disclosure step to display.
  • the public office for example, the Ministry of Health, Welfare, Education, Food and Drug Administration
  • the Internet network remotely billboard It characterized in that it further comprises a hygiene score disclosure step to display.
  • the hygiene score is a score indicating how clean the establishment is. Therefore, guests will choose the restaurant based on the sanitary scores of the billboards posted on the outside.
  • the hygiene score is preferably determined by the presence or absence of detection of target pathogens from food and the frequency of inspections of the establishment.
  • the mobile phone user can receive not only the location information of the restaurant, but also the sanitary score information of the restaurant when searching for a restaurant, which will further contribute to the restaurant user's choice of restaurant and the hygiene of the business.
  • the present invention relates to a DNA extraction disk apparatus using a temperature sensitive material and a DNA extraction method using the same. More particularly, the present invention relates to a cell destruction process, a DNA purification and extraction process, DNA, which are essential for a DNA analysis apparatus. Provided are a DNA extraction disk apparatus for performing DNA analysis more easily and efficiently by integrating an amplification process on one disk, and a DNA extraction method using the same.
  • 3 is another embodiment of a temperature sensitive chamber of a cell disruption chamber
  • 4 (a) and 4 (b) is an embodiment for performing the stirring operation by exerting attractive force and repulsive force on the stirring magnet in the sample stirring chamber for sample homogenization and circulation in the cell destruction chamber,
  • FIG. 5 is an embodiment of a DNA extraction disk apparatus including a drive control unit for driving a DNA extraction disk,
  • 6, 7, 8, and 9 are various embodiments of a DNA extraction disk incorporating a valve, a cell disruption process, a DNA purification process, a DNA extraction process, and a DNA amplification process,
  • FIG. 10 is a cross-sectional view showing an embodiment in which the valve is opened by a capes channel as another embodiment of the valve,
  • the cell disruption chamber 29 is engraved on the disk 100. Feed that may be used in the present invention is injected into the cell disruption chamber 29 through a sample inlet 28.
  • the cell destruction chamber 29 includes a heating film 30 covering the cell destruction chamber 29; A sample stirring chamber 32b; Sample stirring auxiliary chambers 32a and 32c; And a temperature sensitive chamber 16 filled with a temperature sensitive material 24.
  • Reference numeral 44 is a heating device for heating the heating film 30 by a non-contact method, preferably a laser heating device or an induction heating device.
  • the laser heating device 44 is preferably composed of one or more laser modules.
  • the heating film 30 is preferably a metal plate, and the metal plate has a black coating surface 30a coated on one side by black paint and heated by the laser heating device 44. Is preferred.
  • the black coating surface 30a absorbs the light generated from the laser heater 44 to transfer heat to the metal plate 30 to heat the sample in the cell destruction chamber 29.
  • the heating film 30 is preferably a magnetic metal plate
  • the induction heating device 44 sends an alternating current to a coil (not shown), a magnetic force line is generated in the coil, and the magnetic force line faces the coil by Faraday's law of electromagnetic induction.
  • the magnetic metal plate 30 By generating an eddy current in the magnetic metal plate 30 placed in position, the magnetic metal plate 30 is heated to heat the sample in the cell destruction chamber 29.
  • the cell disruption chamber 29 is an enzyme that breaks down proteins, and it is preferable to have an enzyme proteinaseK in the form of a powder in the chamber 29 for activating destruction of the cell membrane.
  • the temperature sensitive chamber 16 is preferably configured in which a chamber for storing a plurality of temperature sensitive materials 24 is arranged in an array form.
  • FIG. 1 shows one embodiment in which six chambers for storing temperature sensitive material 24 are arranged in an array.
  • Reference numeral 170 denotes a disk gap.
  • the temperature sensitive material 24 has a hysteresis phenomenon showing a characteristic difference between a temperature rise and a fall, and this hysteresis phenomenon is proportional to the volume of the temperature sensitive material 24, so that the temperature By dividing the sensitive material 24 into small amounts and arranging them in an array, there is an advantage in that the hysteresis phenomenon can be minimized.
  • the temperature sensitive material 24 is preferably a phase change material, preferably having a melting point of 55 ⁇ 65 °C.
  • the internal temperature of the cell destruction chamber 29 reaches 55-65 ° C., which is the temperature of the activity of the enzyme proteinaseK, cell destruction is most active and phase change in the temperature sensitive chamber 16 is achieved.
  • the phases change from solid to liquid, turning them from opaque white to transparent.
  • the optical sensor 46 installed below the temperature sensitive chamber 16 the temperature in the cell destruction chamber 29 can be measured in a non-contact manner.
  • the heating device 44 is turned on to heat the cell destruction chamber 29.
  • the temperature of the cell destruction chamber 29 is increased to reach the same temperature as the melting point of the phase change material 24. Is activated and the color of the phase change material 24 becomes transparent.
  • the heating device 44 is turned off or the driving voltage of the heating device 44 is adjusted to a low temperature of the cell destruction chamber 29 to 55-65 ° C., which is an appropriate temperature for activating the enzyme proteinaseK. Keep it.
  • the temperature sensitive material 24 is preferably a low-melting alloy, preferably having a melting point of 55-65 ° C.
  • the internal temperature of the cell destruction chamber 29 reaches 55-65 ° C., which is the active temperature of the enzyme proteinaseK
  • cell destruction is most active and low-temperature fusion alloy in the temperature sensitive chamber 16 low-melting alloys change from solid to liquid.
  • the temperature in the cell destruction chamber 29 can be measured in a non-contact manner by observing the liquidity of the low-temperature fusion alloy 24 by the optical sensor 46 installed below the temperature sensitive chamber 16. have.
  • the heating device 44 is turned on to heat the cell destruction chamber 29.
  • the temperature of the cell destruction chamber 29 is increased to reach the same temperature as the melting point of the low temperature fusion alloy 24. Is activated and the low-temperature melting alloy 24 melts into a liquid state.
  • the heating device 44 is turned off or the driving voltage is adjusted low to maintain the temperature of the cell destruction chamber 29 at 55-65 ° C., which is an appropriate temperature for activating the enzyme proteinaseK.
  • the temperature sensitive material 24 is preferably paper coated or dyed with a temperature sensitive fluorescence material.
  • the emitted fluorescence intensity from the paper 24 changes with the rise and fall of the internal temperature of the cell destruction chamber 29.
  • the temperature in the cell destruction chamber 29 can be measured in a non-contact manner.
  • the temperature sensitive fluorescent material 24 is preferably excited by a light emitting diode 48 having a wavelength of 465 nm and the optical sensor 46 has light passing through a wavelength between 550 nm and 625 nm. It is preferred to have a filter (not shown) in front of the optical sensor 46. The optical filter selectively passes a wavelength band belonging to the fluorescence emitted from the temperature sensitive fluorescent material 24.
  • the heating device 44 is turned on to turn on the cell destruction chamber. (29) is heated. In addition, as the heating device 44 heats the cell destruction chamber 29, the temperature of the cell destruction chamber 29 is increased, so that the fluorescence intensity emitted from the paper 24 is the upper limit temperature at which the enzyme proteinaseK is activated. If the temperature is higher than 65 (eg, 65), the heating device 44 may be turned off or the driving voltage may be adjusted to lower the internal temperature of the cell destruction chamber 29 to reduce the activity of the enzyme proteinaseK. It is kept at 55-65 degreeC which is the temperature which enables.
  • the lower limit temperature eg, 55 ° C.
  • the sample stirring chamber 32B is provided with a stirring magnet 31 moving by the magnetic force of the permanent magnet 47a, so that the stirring magnet 31 is rotated as the permanent magnet 47a rotates or moves. By rocking, the sample in the cell destruction chamber 29 is stirred.
  • the sample stirring auxiliary chambers 32a and 32c not only provide a passage through which the sample circulates during the stirring operation of the sample, but also provide a structure to prevent the stirring magnet 31 from being separated from the sample stirring chamber 32b.
  • the low-temperature melting alloy 24 is further provided with a magnetic bead (mangitc bead) to measure the fluidity of the low-temperature melting alloy 24 according to the movement of the permanent magnet 47a by the optical sensor 46 Is preferred.
  • a magnetic bead to measure the fluidity of the low-temperature melting alloy 24 according to the movement of the permanent magnet 47a by the optical sensor 46 Is preferred.
  • the low-temperature melting alloy in the temperature sensitive chamber 16 changes from solid to liquid. Therefore, since the magnetic beads move as the permanent magnet 47a moves, the low-temperature fusion alloy 24 may have fluidity and may be observed by the optical sensor 46.
  • the heating device is turned off, while when the low melting alloy 24 has lost its fluidity, the heating device 44 is turned on or By lowering the driving power to maintain a constant internal temperature of the cell destruction chamber 29 to 55 ⁇ 65 °C.
  • FIG. 2 shows a side view and a perspective view of various embodiments of the cell disruption chamber 29.
  • FIG. 2 (a) and 2 (c) show a case in which the temperature sensitive material 24 is arranged in an array in the temperature sensitive chamber 16, and FIG. 2 (b) shows a temperature on the upper surface of the temperature sensitive chamber 16.
  • FIG. The case where the sensitive material 24 is arrange
  • the temperature sensitive material 24 of FIG. 2 (b) is preferably a phase change material, preferably having a melting point of 55-65 ° C.
  • the internal temperature of the cell destruction chamber 29 reaches 55-65 ° C., which is the temperature of the activity of the enzyme proteinaseK, cell destruction is most active, and an image of the upper surface of the temperature sensitive chamber 16 is formed.
  • the change materials 24 undergo a phase change from solid to liquid to change from opaque white to transparent.
  • the optical sensor 46 installed below the temperature sensitive chamber 16 the temperature in the cell destruction chamber 29 can be measured in a non-contact manner.
  • the heating film 30 is a metal plate, and the metal plate has a black coating surface 30a coated on one side by black paint and is heated by the heating device 44. Is preferred.
  • the heating film 30 is a case where one side of the upper substrate 100a of the disc is implemented by a black coating layer 30 coated with black paint. It is preferred to be heated by the heating device 44.
  • thermometer 3 is another embodiment of the temperature sensitive chamber 16 of the cell destruction chamber 29 or the DNA chamber 50, in which the chamber is configured in the form of a thermometer in the temperature sensitive chamber 16 to form the temperature sensitive material 24. It shows an embodiment of storing the.
  • the temperature sensitive material 24 is preferably a material that changes in volume with temperature change, and a phase change material or a low-melting alloy is preferable. As the temperature rises in the cell destruction chamber 29 by the heating device 44, the temperature sensitive material 24 changes from solid to liquid phase and thus expands in volume.
  • Reference numeral 25a denotes a temperature sensitive channel providing a movement path of the temperature sensitive material 24 according to the volume expansion of the temperature sensitive material 24 due to the temperature rise, and reference numeral 25b denotes a scale indicating the degree of volume expansion.
  • the scale 25b is read by the optical sensor 46 to determine whether the internal temperature of the cell destruction chamber 29 is in the range of 55 to 65 ° C., which is the active temperature of the enzyme proteinaseK, and accordingly the heating Device 44 is feedback controlled.
  • the scale 25b is read by the optical sensor 46 to determine the internal temperature of the DNA chamber 50, whereby the heating device 44 is feedback controlled.
  • Figure 4 (a) and 4 (b) exert an attractive force and reaction force against the stirring magnet 31 in the sample stirring chamber 32b for homogenizing and circulating the sample in the cell destruction chamber 29, thereby stirring operation.
  • Figure 4 (a) is provided with a permanent magnet 47a on the slider 211 to perform the stirring operation, the stirring in accordance with the repetition of the forward and backward movement of the slider 211 With respect to the magnet 31, an embodiment for exerting an attractive force and a reaction force to perform a stirring operation on the sample in the cell destruction chamber 29 is shown.
  • the slider 211 is controlled to move in a radial direction according to the rotation of the slider motor 109 by worm gear connecting portions 109a and 109b connected to the slide motor 109 axis.
  • the slider 211 is moved back and forth to make the stirring magnet 31 swing.
  • the slider 211 is slidably moved using the slide arms 108a and 108b as guides.
  • the slide arms 108a and 108b are fastened to the body of the drive control unit via screws 110a, 110b, 110c and 110d.
  • Reference numeral 113 is a turn table for placing the disk.
  • Another aspect of this embodiment is to observe the transparency of the phase change material on the slider 211, to observe the liquidity (liquidity) of the low-temperature melting alloy, to observe the fluorescence intensity emitted from the paper, or the temperature
  • the volume change of the sensitive material it is characterized in that it is equipped with an optical sensor 46 capable of measuring the temperature in the cell destruction chamber 29 in a non-contact manner.
  • valve opening and closing means 49 for controlling the opening and closing of the valve on the disc is mounted on the slider 211. Space addressing of the valve is possible by adjusting the disk rotation angle and the radial distance of the slider 211.
  • valve opening and closing means 49 is preferably a laser diode, and first performs space addressing on the valve to be opened, and then turns on the laser diode 49 to turn on the valve. Melting and opening are more preferred.
  • FIG. 4 (b) shows a permanent magnet 47a attached to a shaft 300a of the stirring motor 300, so that the stirring magnet 31 in the chamber 32B for sample stirring during the rotation of the stirring motor 300, Exercising the attractive force and the reaction force is an embodiment for performing the stirring operation.
  • FIG. 5 shows an embodiment of the DNA extraction disk apparatus 200 including a drive control unit for driving the DNA extraction disk 100.
  • the driving control unit includes a turn table 113 for placing the DNA extraction disk; A brushless motor 102 for rotating the DNA extraction disk 100 on the turn table 113; And a central control unit 101.
  • a brushless motor suitable for low noise and high speed rotation is preferred as the disk rotation motor.
  • Reference numeral 211 denotes a slider which allows the movement in the radial direction, and the radial movement of the slider 211 is controlled by the step motor 109.
  • Reference numeral 322 is a sanitary score billboard for the central control unit 101 to remotely receive a hygiene score from a public office through an internet connection to display a sanitary score of a business.
  • the hygiene score is preferably displayed on the hygiene score billboard 322 via a wireless connection.
  • Reference numeral 350 denotes a body that supports the DNA extraction disk apparatus 200.
  • a circuit board 140 is jointly fastened to the body 350 at the bottom of the DNA extraction disk device 200, and the central control unit 101, the storage device 112, and the USB and the Internet are connected to the circuit board 140.
  • An input / output device 111 providing a connection is arranged and designed on the circuit board 140.
  • the central controller 101 controls the brushless motor 102 to rotate or stop the disk 100, the heating device 44, the optical sensor 46, the light emitting diode 48, In addition to controlling the valve opening and closing means 49, the stirring motor 300, and the hygiene score billboard 322, the optical sensor 46 and the valve opening and closing designed and arranged on the slider 211 by the control of the slide motor 109. The movement of the means 49 and the permanent magnet 47a is controlled.
  • the central controller 101 controls the display unit 320 and the button input unit 321 to provide a user interface to the DNA extraction disk device 200 to the user.
  • a unique ID of the disc 100 to the central control unit 101 preferably via the wireless RF IC 188 on the disc at the time of loading of the disc to the turntable 113.
  • the central control apparatus 101 recognizes that the DNA extraction disk 100 is loaded by transmitting the information wirelessly.
  • Reference numeral 104 is a crimping means of the disk 100 loaded in the disk cavity 170 is compressed by a magnetic attraction force with the turn table 113 is preferably designed to allow vertical movement and idling.
  • Reference numeral 108 denotes an RF power supply for supplying power to the RF IC 188 by electromagnetic induction.
  • FIG. 6, 7, 8, and 9 illustrate various embodiments of a DNA extraction disk 100 incorporating a cell disruption process, a DNA purification process, a DNA extraction process, and a DNA amplification process.
  • the DNA extraction disk 100 illustrated in FIGS. 6, 7, 8, and 9 includes a sample inlet 28 for injecting a sample; A cell destruction chamber (29) comprising an enzyme and a temperature sensitive chamber (24) for chemically destroying cells contained in the sample while storing the injected sample; The DNA and debris generated from the cells destroyed in the cell destruction chamber 29 are captured by the centrifugal force during the rotation of the disk 100 while the remaining debris is passed through as it is.
  • Silica membrane 51 A membrane chamber 51a on which the silica membrane 51 is fixed; Washing solution chamber 1 (58A) and washing solution storing washing solution 1 (washing solution 1) and washing solution 2 (washing solution 2) for washing the silica membrane (51) capturing the DNA, respectively Chamber 2 58B; Waste chamber for storing debris passing through the silica membrane 51 or for storing impurities generated during the purification and washing of the silica membrane 51 by the cleaning solution 1 and the cleaning solution 2. 52; An extraction solution chamber 59 storing an extraction buffer for extracting DNA bound to the silica membrane 51 washed by the washing solution 1 and the washing solution 2; And a DNA chamber 50 for storing DNA extracted from the silica membrane 51 by the extraction buffer or for amplifying the stored DNA.
  • Another aspect of the invention is to further comprise a porous plastic filter (not shown) in the membrane chamber (51a), it is preferred to mechanically support the silica membrane (51).
  • the porous plastic filter can prevent the silica membrane 51 from deforming mechanically by centrifugal force during the rotation of the disk 100.
  • the disc 100 has valves 56A, 56B, 56C, 56D at the outlet of the cell disruption chamber 29, the wash solution chamber 1 58A, the wash solution chamber 2 58B, and the extract solution chamber 59, respectively.
  • the liquids stored in these chambers are moved along the channel 57 by the centrifugal force generated during the rotation of the disk 100 when the valve is opened.
  • valves 56A, 56B, 56C, 56D, 71b are all initially closed and the valve 71a is opened from the beginning, so that the cell breaking process in the cell destruction chamber 29 is completed by the valve opening and closing means 49
  • the DNA is captured on the silica membrane 51 while the DNA and the debris generated in the cell destruction process are moved along the channel 57 by the centrifugal force according to the rotation of the disk 100.
  • the remaining debris is passed through and stored in the residue chamber 52.
  • valve 56B is opened, and then, by centrifugal force due to the rotation of the disk 100, the washing solution 1 stored in the washing solution chamber 1 58A moves along the channel 57 to capture the DNA. Clean the silica membrane 51. At this time, impurities generated during the cleaning process are stored in the residue chamber 52.
  • valve 56C is opened, and then, by centrifugal force due to the rotation of the disk 100, the washing solution 2 stored in the washing solution chamber 2 58B moves along the channel 57 to capture the DNA. Clean the silica membrane 51. At this time, the impurities generated during the cleaning process are stored in the waste chamber 52.
  • valve 71a is closed by the valve opening and closing means 49, and the valve 71b and the valve 56D are opened.
  • the extraction buffer stored in the extraction solution chamber 59 moves along the channel 57 to bind the DNA bound on the silica membrane 51. Extracted and moved to the DNA chamber 50.
  • reference numerals 71a and 71b denote valves.
  • the valve 71b is initially closed while the valve 71a is open from the beginning so that debris passed as it is without binding to the silica membrane 51 by centrifugal force during the rotation of the disc 100 is left.
  • impurities generated during the washing of the silica membrane 51 by the cleaning solution 1 and the cleaning solution 2 are transferred to the waste chamber 52 and stored.
  • valve 71b and valve 56D are opened while valve 71a is closed.
  • the extraction buffer stored in the extraction solution chamber 59 passes through the silica membrane 51 to extract DNA and transfer the DNA to the DNA chamber 50.
  • reference numeral 77 denotes a Coriolis channel, which is a channel using the Coriolis effect, which is a physical law, and is composed of channels 77a and 77b.
  • the channel 77b and the channel 77a are connected to the outlet of the membrane chamber 51a and branched in opposite directions to form an arch.
  • the channel 77b is connected to the waste chamber 52 and the channel 77a is the DNA.
  • the liquid collected in the membrane chamber 51a is transferred to the DNA chamber 50 via the channel 77a.
  • the debris passed as it is without binding to the silica membrane 51 is transferred to the dreg chamber 52 by centrifugal force during the rotation of the disc 100, or the washing solution 1 and the washing solution 2
  • the disk 100 is rotated counterclockwise.
  • the disk 100 is rotated clockwise.
  • the closing of the valves 56A, 56B, 56C, 56D, 71b is preferably to close the hole of the valve by the black membrane.
  • the black membrane is black vinyl, black polyester film, black paint coated PVDF, black hotmelt, black polyethylene film, black polypropylene, black PVC vinyl (polyvinyl chloride) , Black PET (Polyethylene Terephthalate, Poly-Ethylene-Terephthalate) film and other black synthetic materials are preferred, and the valve opening and closing means is preferably a laser diode.
  • the black membrane has a high absorbance and is easily heated and melted by irradiation of a laser beam by a laser diode to open the valve.
  • the DNA extraction disk 100 illustrated in FIG. 7 is an embodiment in which two pairs of the DNA extraction disk 100 illustrated in FIG. 6 are arranged in a symmetrical structure, and the disk is rotated when the disk 100 is rotated because of the symmetrical structure.
  • the overall weight balance of (100) provides the advantage of minimizing vibration and noise during high-speed rotation of the disk (100).
  • FIG. 9 is an embodiment in which the DNA extraction disk 100 illustrated in FIG. 9 is arranged in a symmetrical structure facing two pairs of the DNA extraction disk 100 illustrated in FIG. 8, when the disk 100 is rotated due to the symmetrical structure.
  • the overall weight balance of the disk 100 provides an advantage of minimizing vibration and noise during high-speed rotation of the disk 100.
  • FIG. 10 is a side view and a plan view of another embodiment in which the valve 71b is opened by a capes channel 72 as another embodiment of the valve 71b.
  • Fig. 10 (a) shows the case where valve 71b is closed by black vinyl 85, while Fig. 10 (b) shows the case where valve 71b is open.
  • the disk 100 is preferably made of an upper substrate 100a, an intermediate substrate 100b and a lower substrate 100c, which are preferably bonded by an adhesive.
  • the top view of FIG. 10 is drawn for the intermediate substrate 100b and the lower substrate 100c, except for the upper substrate 100a.
  • valve 71b is opened by forming a capillary channel 72 on the black vinyl 85 while melting the black vinyl 85 by the valve opening and closing means 49.
  • the liquid in the membrane chamber 51a may move to the DNA chamber 50 by the centrifugal force due to the rotation of the disk 100.
  • the capillary channel (72) extends from the outlet of the membrane chamber (51a) to the hole (71b) of the valve by black vinyl (85) to form a closing channel (closing channel);
  • the valve 71b is formed by turning on a laser diode 49 to melt the black vinyl 85 from the outlet of the membrane chamber 51a to the hole 71b of the valve, thereby forming an opening channel. It characterized in that the opening.
  • the channel 72 is named as a capillary channel.
  • the black vinyl is black polyester film, black hotmelt, black paint coated PVDF, black polyethylene film, black PP, black paint, black PVC vinyl ), Black PET (Polyethylene Terephthalate, Poly-Ethylene-Terephthalate) film and other black synthetic materials are preferred.
  • FIG. 11 is an embodiment of the valve 71a.
  • FIG. 11 (a) shows the case where the valve 71a is opened and
  • FIG. 11 (b) shows the case where the valve 71a is closed.
  • the valve 71a is initially opened as shown in FIG. 11A, so that the liquid collected in the membrane chamber 51a by centrifugal force can freely move to the dreg chamber 52.
  • the disk 100 is preferably made of an upper substrate 100a, an intermediate substrate 100b and a lower substrate 100c, which are preferably bonded by an adhesive.
  • the top view of FIG. 11 is drawn for the intermediate substrate 100b and the lower substrate 100c, except for the upper substrate 100a.
  • Reference numeral 78b denotes a PCM chamber which stores a phase change material 78, and when heated by the laser diode 49, the phase change material 78 changes from solid to liquid.
  • Reference numeral 79 denotes a black coating surface made of black paint, which is present on the bottom surface of the phase change material 78 so that when the laser diode 49 is turned on, the light emitted from the laser diode 49 is highly efficient. Can absorb. Accordingly, the black coating surface 79 is heated, and the phase change material 78 melts by this heat, and the phase change material 78 flows into the channel, thereby closing the valve 71a as shown in FIG. 11 (b). . After the phase change material 78 enters the channel, the laser diode is turned off, causing the phase change material 78 to turn into a solid state so that the valve 71a remains closed.

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Abstract

La présente invention concerne : un appareil à disque d'extraction d'ADN, un matériau sensible à la température étant positionné dans une chambre de désintégration cellulaire pour mesurer directement la température d'un échantillon dans la chambre et réguler ainsi la température dans la chambre de désintégration cellulaire par la commande de rétroaction de la marche/l'arrêt d'un dispositif de chauffage, et ainsi, l'appareil à disque d'extraction d'ADN effectue chimiquement une procédure de désintégration cellulaire sur l'échantillon pour purifier et extraire l'ADN intracellulaire ou amplifier l'ADN extrait ; et un procédé d'extraction d'ADN mettant en œuvre ce dernier.
PCT/KR2016/001318 2015-02-05 2016-02-05 Appareil à disque d'extraction d'adn et procédé d'extraction d'adn mettant en œuvre ce dernier Ceased WO2016126141A1 (fr)

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US20230001409A1 (en) * 2019-11-18 2023-01-05 Bl Technologies, Inc. Bacterial endotoxin reader verification plates and methods of use

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SG11201903252RA (en) * 2016-10-11 2019-05-30 Haemokinesis Pty Ltd Method for enhancing the incubation of samples, specimens and reagents using lasers
KR101967236B1 (ko) * 2017-05-16 2019-04-09 에스케이텔레콤 주식회사 핵산 추출용 전처리 챔버, 그를 이용한 카트리지 및 핵산 추출 방법
KR102013698B1 (ko) * 2017-12-20 2019-08-26 주식회사 씨디젠 검정색 열가소성 수지 밸브 및 비접촉 온도 센서를 이용한 랩온어 디스크 장치
KR102300539B1 (ko) * 2021-05-04 2021-09-09 주식회사 에이아이바이오틱스 Rt-pcr 측정용 인터페이스 모듈 및 이를 포함하는 회전형 rt-pcr 디바이스

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