WO2003080829A1 - Dispositif de piegeage/liberation d'adn utilisant un canal, et procede de piegeage et de liberation d'adn - Google Patents

Dispositif de piegeage/liberation d'adn utilisant un canal, et procede de piegeage et de liberation d'adn Download PDF

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WO2003080829A1
WO2003080829A1 PCT/JP2003/003747 JP0303747W WO03080829A1 WO 2003080829 A1 WO2003080829 A1 WO 2003080829A1 JP 0303747 W JP0303747 W JP 0303747W WO 03080829 A1 WO03080829 A1 WO 03080829A1
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Prior art keywords
dna
liquid
charged linear
electric field
pressure difference
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PCT/JP2003/003747
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English (en)
Japanese (ja)
Inventor
Jun Kikuchi
Yasuhiro Horiike
Yuzuru Takamura
Tetsuya Hayama
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Jun Kikuchi
Yasuhiro Horiike
Yuzuru Takamura
Tetsuya Hayama
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Application filed by Jun Kikuchi, Yasuhiro Horiike, Yuzuru Takamura, Tetsuya Hayama filed Critical Jun Kikuchi
Priority to AU2003227224A priority Critical patent/AU2003227224A1/en
Publication of WO2003080829A1 publication Critical patent/WO2003080829A1/fr

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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
    • 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
    • B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • 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
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0663—Stretching or orienting elongated molecules or particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

Definitions

  • the present invention is intended to separate and separate only leukocytes, pathogenic bacteria, and viral DNA contained in blood, etc. in order to judge health conditions, perform early detection of infectious diseases, early treatment, and tests during treatment.
  • the present invention relates to a separation means for extracting, concentrating, and performing a diagnosis based on DNA information, and an apparatus therefor.
  • a part of the necessary functions and structures are integrated on a single plate-shaped chip, and only a small amount of the required sample is required.
  • Leukocytes and somatic cells provide information on the individual's genetic information, which is important for diagnosis of lifestyle-related diseases and genetic diseases, and for personalized medicine.
  • infectious diseases can be diagnosed by extracting and specifying DNA of pathogenic bacteria and viruses contained in blood and the like.
  • high-sensitivity selection can be achieved by combining it with a method that amplifies only DNA with a specific sequence using a DNA chip, PCR, or LAMP.
  • Highly sensitive detection is possible in principle.
  • Preparing DNA and salt concentrations and pH suitable for PCR and LAMP methods is required by removing the DNA that disrupts the membrane and virus wall and removing substances that would hinder subsequent reactions and detection. It is.
  • Pretreatment consists of repeating the mixing, reaction, and recovery of DNA. Examples of the form of the recovery treatment include concentration and separation of DNA, exchange of a solution that leaves only DNA, and washing of DNA by exchanging the solution.
  • Centrifugation which is used for DNA recovery, relies on manual separation of supernatant, intermediate layer, and sediment, making it difficult to apply to small samples It is.
  • the technical fields of micro TAS and Lab-on-a-Chip have been emerging, and by integrating conventional analytical techniques and chemical synthesis methods on a single chip, the system can be made smaller, The required reagent and sample volume have been reduced, cost has been reduced, speed has been improved, and functions have been improved.
  • DNA analysis can be expected from a drop of blood, several cells or viruses.
  • DNA recovery technology that can be realized in a chip is required.
  • the method of entanglement with magnetic beads can be realized on a chip, but the movement of the magnetic field is complicated, and the collected DNA must be released again into the solution so that the next processing can be performed (ie, release). ) Also lacks generality, such as not being released with a simple buffer.
  • DNA pretreatment for diagnostic purposes has not yet been able to concentrate to a level that allows detection of the antigenic blood virus. At the cell and bacterial level, various techniques have been developed, but they are cumbersome, unsuitable for a complete chip, or sacrificing sensitivity.
  • a chemical kit has been developed that completes pretreatment simply by mixing it with a solution containing cells without concentrating or recovering DNA. However, compared to the purification method, the sensitivity and accuracy are higher. Is bad.
  • FIG. 1 is an illustration of means for solving the problem.
  • a solution containing DNA 102 is passed through the channel 101.
  • a pressure difference and an electric field are simultaneously applied to the solution. That is, in the DNA 102 in the solution, the force in the direction indicated by the direction 103 due to the pressure flow and the force in the direction indicated by the direction 104 due to the electric field are shown in Fig. 1.
  • the flow channel 101 has one or more wide portions 105 and one or more narrow portions 106.
  • the DNA 102 can be selectively placed near the narrow portion by adjusting the strength of the electric field and the pressure flow. Can be trapped. By adjusting the strength of the electric field and the pressure, the flowing DNA can be trapped one after another. Substances in the solution other than those trapped at this time flow off due to the electric field or pressure flow, so that DNA recovery and concentration can be realized. Since the solution is flowing while the DNA is trapped, it can be used for solution exchange and washing.
  • the narrow part has a cross-sectional area from 0.01 micron to 50 microns, and the wide part has more than twice the cross-sectional area of the narrow part.
  • the trap capacity of the present invention depends on the size of the DNA. It is possible to change the size of the trappable DNA by changing the channel diameter and shape, or by changing the voltage and pressure.
  • FIG. 1 is a diagram of a trapping mechanism according to the present invention
  • FIG. 2 is a basic diagram of an embodiment utilizing the present invention
  • FIG. 3 is a diagram of an application example of an embodiment of the present invention.
  • FIG. 4 is a diagram of a configuration example of a trapping mechanism of the present invention
  • FIG. 5 is a diagram of a method of arranging a plurality of trapping mechanisms of the present invention to achieve an effect
  • FIG. 7 is a diagram of a method of performing separation by connecting the present invention having different effects in series
  • Fig. 7 is a diagram of a configuration for analyzing blood or the like using the present invention
  • Fig. 8 is a diagram of the present invention.
  • FIG. 9 is a diagram of an example of an apparatus configuration for carrying out the present invention
  • FIG. 9 is a diagram of the migration speed of DNA only in the pressure field in the embodiment
  • FIG. 10 is a diagram of the migration of DNA only in the electric field in the embodiment.
  • Fig. 11 is a diagram of velocity.
  • Fig. 11 shows trapping of DNA when both a pressure field and an electric field are applied in the embodiment.
  • Fig. 12 is a diagram of the range of voltage and pressure at which trapping occurs in the embodiment
  • FIG. 13 is a diagram of the range of voltage and pressure at which trapping occurs in reverse connection in the embodiment.
  • Fig. 14 is a diagram of the relationship between the moving distance of DNA and time when both a pressure field and an electric field are applied in the embodiment.
  • Fig. 15 is a diagram of the DNA when only the pressure field in the embodiment is used.
  • FIG. 16 is a diagram of the relationship between the moving distance and time of the DNA, and FIG. 16 is a diagram of the relationship between the moving distance of DNA and the time when only the electric field is used in the embodiment.
  • FIG. 17 is a diagram of the current tracking phenomenon.
  • FIG. 18 is an explanatory view of an application example in which a liquid supply device is added to the present invention. BEST MODE FOR CARRYING OUT THE INVENTION
  • FIG. 2 shows the most basic form of trapping and releasing DNA based on the present invention.
  • the trap mechanism 201 is a flow path according to the present invention having a wide portion and a narrow portion.
  • Entrance (reservoir) 202 is an entrance for DNA, into which a liquid containing DNA is put. The liquid flows through the flow path 204, the trap mechanism 201, and the outlet ( ⁇ esthetic) 203.
  • Entrance 2 0 2 and Pressure is applied using a tube from either or both outlets 203 to a pressure source.
  • An electric field is simultaneously applied to the entrance 202 and the exit 203 using electrodes or the like.
  • the liquid is transported from the inlet 202 to the outlet 203 by pressure, and at the same time, a positive voltage is applied to the inlet 202 and a negative voltage is applied to the outlet 203.
  • a positive voltage is applied to the inlet 202 and a negative voltage is applied to the outlet 203.
  • only the DNA in the liquid at the entrance 202 is trapped by the trap mechanism 201.
  • the concentrated DNA can be taken out at the entrance 202 side.
  • the concentrated DNA can be taken out at the outlet 203 side.
  • FIG. 3 shows an embodiment of the present invention.
  • a DNA recovery port 302 serving as a concentrated DNA recovery channel 302 and a reservoir for adding an electrode is provided at the entrance 301 side.
  • 303 is provided.
  • DNA is caused to flow from the entrance 301 to the trap mechanism 201 by pressure, and a positive voltage is applied to the DNA recovery opening 303 and a negative voltage is applied to the exit 203.
  • the concentrated DNA can be recovered to the DNA recovery channel 302 and the DNA recovery port 303 via the channel 204. Even if the recovery path is on the exit side of 203, recovery can be performed in the same manner.
  • the lower diagram in FIG. 3 shows a use mode of the present invention, in which a detection unit 304 is attached at the end of the DNA recovery channel 302.
  • the detection unit 304 may be a PCR, a LAMP champer, or a DNA chip.
  • various electrophoresis rams may be used. If electrophoresis is to be performed, the sample must be plugged into a starting point at the beginning of electrophoresis. Usually, this is realized using a crossroad or the like. However, since the DNA released according to the present invention is released in the form of a plug, it may be omitted. Also detect The part 304 is replaced by an element that performs post-processing other than detection.
  • FIG. 18 shows a configuration obtained by adding a liquid supply device 1801 to the configuration shown in the lower part of FIG.
  • the liquid containing DNA is conveyed from the inlet 301 to the outlet 203 by pressure, and a positive voltage is applied to the inlet 301 and a negative voltage is applied to the outlet 203 at the same time.
  • a positive voltage is applied to the inlet 301 and a negative voltage is applied to the outlet 203 at the same time.
  • the trap mechanism 201 only the DNA is trapped by the trap mechanism 201.
  • supply of liquid from the inlet 301 is stopped while the force received by the DNA from the pressure flow and the force received from the electric field are maintained, and the liquid is replaced from the liquid supply device 1801
  • the exchange liquid stored in the liquid reservoir 1802 By supplying the exchange liquid stored in the liquid reservoir 1802, the liquid around the DNA can be exchanged while the DNA is trapped.
  • the exchange liquid by continuously flowing the exchange liquid for a required time, it is possible to treat the DNA with the exchange liquid and to wash the DNA. Then, by increasing the electric field, or decreasing, turning off or reversing the pressure, the liquid can be exchanged, the treated or washed DNA can be recovered, and the next treatment can be similarly performed.
  • FIG. 4 shows various forms for realizing a narrow channel width portion and a wide channel width portion of the channel 101 constituting the trap mechanism of the present invention.
  • the upper diagram simply shows different widths. This is realized by connecting the flow paths. The width may be in the horizontal direction of the chip, in the depth direction, or both.
  • a trap mechanism is formed by filling the flow path wall 401 with obstacles 402 such as beads.
  • the lower part of FIG. 4 is an example in which this is realized by forming a porous material 403 such as a porous film or a resin in the flow path wall 401. It can be easily realized by using photo-curing resin.
  • Fig. 5 shows a configuration for connecting multiple trap mechanisms in parallel.
  • Reference numeral 01 denotes an inlet of the flow channel
  • 502 denotes an outlet of the flow channel, which can improve throughput and trap amount.
  • the upper two are forms of multiple integration on a plane. Since the trapping mechanism is very small, a capacity improvement of about 1000 times can be easily obtained by integration.
  • the lower part realizes a trapping mechanism by the holes formed in the plate-like material and the space adjacent to the holes, so that a higher degree of integration can be easily obtained.
  • Fig. 6 shows a configuration in which trap mechanisms 601 of different sizes that can be trapped are connected in series, directly or without a connection path 622, so that different sizes of DNA can be trapped. Can be trapped. This makes it easy to separate by size and can be applied to analysis and DNA filtering.
  • the collection path 603 is connected to the connection path 601, it is possible to collect and collect different DNAs for each size or to perform post-processing.
  • Fig. 7 shows the configuration of a DNA analyzer using this mechanism.
  • the virus wall is disrupted.
  • the DNA trap is then injected into the DNA trap mechanism 703 using the various forms of the present invention described above, and only the DNA is concentrated and recovered from the disrupted cell wall, white matter, and ions. I do.
  • the released DNA is selectively detected by the detector 704.
  • the detection unit 704 may be a PCR method, a LAMP method, an electrophoretic force ram, or the like.
  • FIG. 8 shows one example of trapping DNA using the present invention.
  • DNA801 stained with YOY01 can be observed with a fluorescence microscope It is.
  • T 4-D NA size 160 k BP
  • ⁇ -D NA size 48 k BP
  • DNA is contained in 0.5 TBE buffer, along with mercaptoethanol, glucose oxidase, catalysin, and darcos, to prevent DNA from being cut during observation by dissolved oxygen.
  • a voltage is applied by the power supply 802.
  • the pressure gauge 803 measures the applied pressure using a syringe 804. Apply voltage to the entrance and exit using platinum wire 805.
  • Reference numeral 806 is a glass plate, 807 is silicone rubber, and both are used to seal the entrance and the exit.
  • Reference numeral 808 denotes a quartz chip, and a trap mechanism such as a continuous wedge type as shown in FIG. 1 is arranged as shown in FIG. The thinnest part of the wedge is 0.6 microns, the thick part is 5 microns, the period of the wedge is 50 microns, and the number of repetitions is eight. The depth is 0.5 microns.
  • Reference numeral 809 denotes a lens for observing the state of DNA in an enlarged manner. 810 is a dichroic mirror, 811 is a mirror, 813 is excitation light (490 nm), and is an optical system for fluorescence observation. The fluorescence 8 14 from the DNA is observed with a high-sensitivity CCD camera 8 12.
  • Fig. 9 shows an example in which DNA was first electrophoresed by applying only pressure. The figure shows that the pressure difference was about 40 Pa or less, but there was a difference in speed. Neither the large DNA (T 4) nor the small DNA (the fragment) was trapped, even by difference. As the pressure differential increased, DNA passed through this channel more easily. This was the same even if the wedge direction was reversed. This can be used for the release after the trap of the DNA described later.
  • FIG. 10 shows an example in which DNA is electrophoresed by applying only an electric field. This figure is In each case, the voltage was low (less than 0.4), but DNA easily passed through this channel. With a larger voltage difference, it passed through this channel more easily. This was the same even if the direction of the wedge was reversed. This can be used for the release of the trapped DNA described below.
  • Fig. 11 shows that a voltage of 6 V is applied so that the force exerted on the DNA by the electric field becomes 1101 in the direction of the wedge and the direction of the force in the pressure flow 1 1
  • a pressure of 5 kPa is applied to 02.
  • the T4-DNA was trapped at the position indicated by DNA 102 in the figure, and did not move for a long time of about 10 minutes. The trapped DNA was released as soon as the electric field was turned off, in the direction of the force from the pressure flow.
  • FIG. 12 shows the condition range of the voltage and pressure at which trapping of T4 and DNA in FIG. 11 occurs.
  • Reference numeral 1201 denotes a trapping range (shaded area). Under the conditions in this range, T4-DNA was reliably trapped. In the region above approximately 3 kPa, traps can be seen near the voltage that balances the pressure flow. The range of voltages at which trapping occurred widened as pressure increased. In addition, DNA smaller than T4 DNA was released slightly inside this condition, indicating that the trap was size-dependent.
  • Fig. 13 shows the range in which traps occur when the pressure and voltage directions in Fig. 11 are both reversed. As in Figure 12, as the pressure increased, the voltage trapping range became wider.
  • FIGS. 12 and 13 show the conditions under which the force from the pressure flow and the force from the electric field exert on DNA are balanced. Therefore, in this trap, the force due to pressure and the force due to the electric field balance in the DNA. It occurs in the range of 30% to 170% of the condition, indicating that it is necessary to apply a force higher than a certain threshold determined by the shape and the DNA molecule.
  • FIG. 14 shows the movement of large and small DNAs below the trap region in FIG. From this figure, it was found that in such a shape, when both the pressure and the electric field were applied in opposite directions, the migration speed was significantly size-dependent. Similar remarkable size dependence was observed above the trap region. This is a DNA size separation method that replaces electrophoresis using gels or capillaries.
  • Figs. 15 and 16 show plots similar to Fig. 14 when only the pressure and electric fields are used. It turns out that there is almost no.
  • the trap was performed by changing the size of the narrowest part and the wide part of the wedge type. It was found that the wide part can be trapped without problem even if it exceeds 100 microns, which is considered to be sufficiently infinite from the size of the trapped DNA.
  • the size of DNA that can be trapped changes even at the same pressure and electric field.At 0.6 micron, DNA of 100 Obp or more is trapped, and at 0.3 micron, 50 Obp is trapped. The above DNA was trapped. In addition, no trap of DNA was observed in the case of 50 micron. From this result, it is better to trap the narrowest part to trap small DNA, and to optimize the size of the narrow part, the voltage and the pressure It turns out that DNA can be trapped. Also, the width of the narrow portion effective for trapping the DNA is considered to be between 0.11 micron and 50 microns, considering the size of the DNA.
  • the fluorescence observation shown in FIG. Use a monitoring device.
  • the electrophoresis chip 808 has an inlet 301 and an outlet 203, a DNA recovery channel 302, a DNA recovery port 303, and a liquid supply port.
  • the trap mechanism 201 has a wedge thinnest of 0.6 microns, a thick part of 5 microns, a wedge cycle of 50 microns, and a repetition of eight times, as in the first embodiment. .
  • the depth is 0.5 micron.
  • Buffer A 0.5 TBE buffer with mercaptoethanol, dalcosoxidase, catalase, and glucose to prevent DNA breakage during observation due to dissolved oxygen, and polyvinylpyrrolidone to suppress electroosmotic flow
  • buffer A A solution obtained by mixing polystyrene beads having a CO ⁇ H group on the surface thereof with a DNA solution prepared in the same manner as in Example 1 is put into the inlet 301.
  • a syringe for applying pressure and a platinum electrode are connected to the entrance 301 in the same manner as in the first embodiment.
  • a platinum electrode is connected to the outlet 203.
  • a syringe for applying pressure is connected to each of the exchange solution reservoir 1802 and the DNA recovery column 303. These four connections are each sealed with silicone rubber as in the first embodiment.
  • a compression pressure of 8 kPa is applied to the inlet 301, and at the same time, a voltage of 10 V is applied between the outlet 203 and the inlet 301 so that the outlet 203 becomes negative.
  • the pressure applied to the exchange solution reservoir 1802 and the DNA recovery column 303 is adjusted so that DNA does not flow in the liquid supply device 1801 and the DNA recovery channel.
  • the DNA, solution, and polystyrene beads at the entrance 301 entered the trap mechanism 201 one after another, and the DNA was trapped and concentrated, but the beads flowed to the exit 203.
  • Figure 17 illustrates the trapping force.
  • 1701 is the force by the electric field, and the force which the DNA receives from the electric field is constant regardless of the distance from the wall.
  • 1702 is the force due to the pressure flow, and the force that the DNA receives from the pressure flow is small near the wall surface and large at the center. Therefore, near the narrowest part, the force due to the electric field is strong near the wall surface, and in the center 1703, a force due to a very strong pressure is generated.
  • Particles flowing here can slip through the wall, but long molecules such as DNA will escape in the process of slipping and somewhere in the middle of the long molecule such as DNA1774 And returned to the trap section. As a result, only long molecules are trapped.
  • the DNA trap mechanism of the present invention Only longer molecules, including DNA or DNA, can be trapped and released as needed. This allows a mechanism to recover only the DNA from the pretreatment solution for extracting the DNA on the chip, an operation to replace the buffer and the solution while leaving the DNA, or a method to concentrate the extremely diluted DNA and perform PCR or the like. This makes it easier to increase the detection sensitivity of the LAMP method, DNA chip, etc., and to adjust the liquid volume to be easily handled on the chip. This facilitates the analysis and diagnosis of leukocytes in the blood, DNA of viruses and pathogens, and has the effect of making antigen diagnosis rather than antibody detection faster and more accurate.
  • new DNA separation methods, DNA filtering methods, and screening methods can be easily configured using the trapping mechanism and size dependence of migration speed.
  • the ability to temporarily fix only DNA in a solution requires the exchange of a solution or the mapping of DNA. It facilitates observation of the duplication and DNA, and can have all kinds of ripple effects.
  • the present invention has been described specifically for DNA, it can be easily presumed that the present invention can be applied to RNA and long-chain linear molecules having similar characteristics. Industrial applicability
  • the trap / release device can selectively trap DNA or charged linear molecules from a solution containing inclusions and release them by a simple operation. Therefore, it is useful for concentrating and extracting specific molecules, exchanging solvents, and washing molecules, and is particularly suitable for pretreatment of removing DNA and RNA from cells on a chip.

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Abstract

On fait passer un liquide contenant de l'ADN à travers un canal comportant une partie large et une partie étroite, grâce à une différence de pression, en appliquant simultanément un champ électrique dans une direction telle que l'ADN s'écoule dans la direction opposée à celle de l'écoulement produit par la différence de pression. Ce système permet de piéger de manière sélective l'ADN à proximité de la partie étroite. L'ADN qui s'écoule peut être piégé séquentiellement grâce à la régulation du niveau du champ électrique et de la pression, ce qui permet de récupérer et de concentrer l'ADN. L'ADN piégé est déchargé immédiatement en direction d'un orifice d'entrée ou de sortie, grâce à l'augmentation ou à la diminution du niveau du champ électrique ou de la pression ; ce système permet de libérer et de récupérer facilement l'ADN en vue d'une utilisation de celui-ci dans un processus ultérieur.
PCT/JP2003/003747 2002-03-26 2003-03-26 Dispositif de piegeage/liberation d'adn utilisant un canal, et procede de piegeage et de liberation d'adn WO2003080829A1 (fr)

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AU2003227224A AU2003227224A1 (en) 2002-03-26 2003-03-26 Dna trap/release apparatus using channel and method of trapping and releasing dna

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JP2002-130183 2002-03-26
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JP2003124520A JP4234486B2 (ja) 2002-03-26 2003-03-26 Ｄｎａ或いは電荷をもつ線状の分子のトラップ・リリース装置とその方法

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JP2005278418A (ja) * 2004-03-26 2005-10-13 Japan Science & Technology Agency 試料から荷電物質を濃縮及び／又は抽出する方法及びそのためのデバイス
JP4692200B2 (ja) * 2005-10-06 2011-06-01 横河電機株式会社 化学処理用カートリッジおよびその使用方法
WO2007055165A1 (fr) * 2005-11-11 2007-05-18 Konica Minolta Medical & Graphic, Inc. Procede de separation d'acide nucleique, microreacteur et systeme d'essai d'acide nucleique
JP2008116318A (ja) * 2006-11-03 2008-05-22 Japan Advanced Institute Of Science & Technology Hokuriku 検体の捕捉方法
WO2008075501A1 (fr) 2006-12-19 2008-06-26 Konica Minolta Medical & Graphic, Inc. Récipient rotatif d'extraction, procédé destiné à identifier des espèces cellulaires et procédé de détection de gène utilisant ledit récipient, et extracteur automatique d'acide nucléique
JP4908182B2 (ja) * 2006-12-22 2012-04-04 栄研化学株式会社 核酸増幅の有無の判定方法、標的核酸の検出方法及びそれらに用いられる装置
WO2013119765A1 (fr) * 2012-02-10 2013-08-15 The University Of North Carolina At Chapel Hill Dispositifs à nano-entonnoirs fluidiques, procédés associés, systèmes de fabrication et d'analyse
US10022728B2 (en) * 2013-02-01 2018-07-17 Arizona Board Of Regents On Behalf Of Arizona State University Punctuated microgradients for improved separations of molecules and particles
FR3024544B1 (fr) 2014-08-01 2019-06-21 Centre National De La Recherche Scientifique Procede et dispositif de concentration de molecules ou objets dissous en solution.
JP2021061767A (ja) * 2019-10-11 2021-04-22 株式会社日立製作所 生体高分子抽出装置及び生体高分子抽出方法

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