WO2003080829A1 - Dna trap/release apparatus using channel and method of trapping and releasing dna - Google Patents

Dna trap/release apparatus using channel and method of trapping and releasing dna Download PDF

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Publication number
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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French (fr)
Japanese (ja)
Inventor
Jun Kikuchi
Yasuhiro Horiike
Yuzuru Takamura
Tetsuya Hayama
Original Assignee
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/en

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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

A liquid containing DNAs is passed through a channel having large width part and small width part by a pressure difference, and simultaneously an electric field is applied in the direction such that DNAs flow in the direction opposite to the pressure difference flow. This enables selectively trapping DNAs in the vicinity of small width part. Flowing DNAs can be trapped in sequence by regulating the level of electric field and pressure, thereby realizing the recovery and concentration of DNAs. Trapped DNAs are discharged at a stretch toward an inlet or outlet by increasing or decreasing the level of either electric field or pressure. This enables easy release and recovery so as to attain use in the subsequent processing.

Description

明 細 書 流路を用いた DNAのトラップ · リ リース装置ならびに D NAのトラッ プ · リ リース方法 技術分野  Description Trap / release device for DNA using flow channel and trap / release method for DNA
本発明は、 健康状態の判断や、 感染症の早期発見、 早期治療、 及び治 療中の検査を行うために、 血液等に含まれる、 白血球、 病原菌、 ウィル スの D NAのみを、 分離、 抽出、 濃縮し、 DN Aの情報に基づく診断を 行うための分離手段ならびにその装置に関する。 特に必要な機能、 構造 の一部が一つの板状のチップに集積されており、 必要な検体が微量です み、 携帯性、 即時性、 使い捨て、 安価などを特徴とする微小流体力学や m i c r o—TA S、 L a b— o n— a— C h i pといわれる分野に関 する。 背景技術  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. In particular, 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. TA S, Lab—On—a—Chip related. Background art
人体から取り出した、 組織や血液等に含まれる D N Aを分析する技術 は非常に重要である。 白血球や体細胞からは本人の遺伝子情報が判り、 生活習慣病や遺伝病の診断、 オーダーメード医療に重要である。 また、 血液等に含まれる病原菌やウィルスの D NAを取り出して特定すること により、 感染症の診断ができる。  Techniques for analyzing DNA contained in tissues, blood, etc. extracted from the human body are very important. 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. In addition, infectious diseases can be diagnosed by extracting and specifying DNA of pathogenic bacteria and viruses contained in blood and the like.
精製された D N Aを特定する方法としては、 D NAチップや、 P C R 法、 L AM P法を用いて特定配列を持った D NAだけを増幅するような 手法と組みあわせることにより、 高感度で選択性の高い検出が原理的に 可能である。 このような検出方を用いるには、 採取した細胞やウィルス が含まれているであろう液体から、 D N Aに有害な物質を除外し、 細胞 膜やウィルス壁を破砕して D NAを取り出し、 その後の反応や検出を阻 害するような物質を除外し、 P C Rや L AMP法に適当な DNA濃度、 塩濃度、 P Hを調整する前処理が必要である。 前処理は試薬の混合、 反 応、 D NAの回収処理の繰り返しからなる。 この回収処理の形態として は、 D NAの濃縮、 分離、 DN Aのみを残した溶液の交換、 溶液の交換 による DNAの洗浄などがある。 即ち、 介在物が存在する溶液から、 で きるだけ選択的に DNAのみを一箇所に集め、 他の介在物と分けて回収 することが必要であり、 また DN Aを 1 まとめにしたまま、 溶液内を移 動する、 あるいは逆に溶液の方を移動することにより、 溶液の交換や洗 浄が実現できれば、 より簡単に前処理を実現できる。 このような DNA の回収を行う方法としては、 遠心分離法、 磁気ビーズを用いて DN Aを 絡め取った磁気ビーズを磁石により移動する方法等がある。 As a method for identifying purified DNA, 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. To use this type of detection, remove harmful substances to DNA from the collected cells or fluids that may contain viruses, 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. In other words, it is necessary to collect only the DNA from the solution containing the inclusions as selectively as possible at one place and collect it separately from the other inclusions. If the solution can be replaced or washed by moving inside the solution or conversely by moving the solution, pretreatment can be realized more easily. As a method for recovering such DNA, there are a centrifugal separation method, a method in which magnetic beads with DNA entangled using magnetic beads are moved by a magnet, and the like.
一方で、現在ウィルス性の病気の診断には、ウィルスに免疫が応対し、 産出された抗体を検出する方法がとられている。 これは、 血液中のウイ ルスが余りに微量なため、 検出が困難であるからである。 しかし、 この 方法には、 感染から免疫が応答し抗体ができるまでの期間、 診断ができ ないという欠点がある。 また、 B型肝炎など、 ウィルスが除去されても 抗体だけ残ったり、 また重症患者では、 新たな感染があっても、 抗体を 産出できず、 誤診される欠点があった。 もし、 血液中の微量なウィルス 由来の D N Aを濃縮することができれば、 これら抗原の直接検出でき、 早期発見、 早期治療に大きく貢献する。 現在、 石英など特定の材料の表 面に D N Aを固定し洗い流す方法等を用いて濃縮が試みられているが、 この要求レベルを満たすようなものはまだない。  On the other hand, for the diagnosis of viral diseases, a method is currently used in which immunity responds to the virus and the produced antibodies are detected. This is because the amount of virus in blood is too small to detect. However, this method has the disadvantage that no diagnosis can be made from the time of infection until the immune response and antibody production. In addition, even if the virus is removed, such as hepatitis B, only the antibody remains. In severely ill patients, even if there is a new infection, the antibody cannot be produced, and the patient is misdiagnosed. If a small amount of DNA derived from virus in the blood can be concentrated, these antigens can be detected directly, which will greatly contribute to early detection and early treatment. At present, there is an attempt to concentrate DNA using a method of fixing and washing away DNA on the surface of a specific material such as quartz, but there is no material that meets this required level.
D N Aの回収処理で用いられる、 遠心分離は、 手による上澄みや中間 層、 沈殿物の取り分け作業に依存しており、 微量なサンプルに適用困難 である。 近年、 m i c r o TAS、 L a b— o n— a— C h i pという 技術分野が立ち上がりつつあり、 従来の分析技術や化学合成法を、 一つ のチップの上に集積化することで、 システムの小型化、 必要な試薬ゃ検 体量の縮小化、 低コスト化、 高速化、 高機能化を実現している。 この技 術を用いると、 一滴の血液、 数個の細胞やウィルスから D N A分析が可 能と期待される。しかしながらこれを実現するには、遠心分離に代わり、 チップの中で実現できる D N Aの回収技術が必要である。 磁気ビーズに 絡め取る方法は、 チップ上でも実現可能であるが、 磁場の移動は煩雑で あり、 また集めた D NAを、 次の処理が行えるように再び溶液中に解放 すること（即ちリ リース） も、単純な緩衝液ではリ リ一スされないなど、 一般性に欠ける。 診断を目的とした、 DNAの前処理では、 前に述べた ように抗原である血中ウィルスを検出できるレベルの濃縮が可能なもの はまだない。 細胞や細菌レベルでは、 いろいろな手法が開発されている が、 煩雑で、 完全チップ化に不向きであったり、 感度が犠牲になったり する。 また、 D N Aの濃縮や回収を行わず、 細胞が含まれている溶液に 混ぜるだけで前処理が完了するような薬品のキッ トが開発されているが， 精製する方法に比較すると、 感度や精度は悪い。 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. In recent years, 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. With this technology, DNA analysis can be expected from a drop of blood, several cells or viruses. However, to achieve this, instead of centrifugation, 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. As mentioned earlier, 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. In addition, 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.
これらの要求に答えるべく、チップ上に形成した簡単な機構を用いて、 流路を流れるたんぱく質やさまざまな試薬類を含む液体から、 DNAあ るいはそれに類するものを選択的に捕捉保持 （即ち トラップ） し、 DN Aの回収、 或いは D N Aの濃縮を行い、 またトラップされた D N Aを簡 単な操作で、 リ リースし、 次の処理に用いることができるような、 安価 で単純な原理 · 装置はまだない。 発明の開示 図 1は課題を解決する手段の説明である。 流路 1 0 1の中に DNA 1 0 2を含む溶液を流す。 溶液には、 圧力差と電界を同時に印加する。 即 ち、 溶液中の D N A 1 0 2には、 圧力流れによる力の方向 1 0 3で示さ れる方向の力と、 電界による力の方向 1 04で示される方向の力が、 図 1に示されるように互いに逆向きになるように同時に働かせる。 この 2 つの力の向きは互いに逆向きであれば反対方向でも良い。 流路 1 0 1に は、 一つ以上の流路幅の広い部分 1 0 5 と、 一つ以上の流路幅の狭い部 分 1 0 6が存在する。 流路 1 0 1の具体的な形状は後述するように様々 であるが、電界と圧力流れの強さを調節することにより DNA 1 0 2を、 選択的に、 幅が狭い部分の近辺に、 トラップすることができる。 また電 界ゃ圧力の強さを調節することにより、 流れてきた DNAを次々にトラ ップすることができる。 この時トラップされるもの以外の溶液中の物質 は、 電界あるいは圧力流れにより流れ去るので、 DNAの回収や濃縮が 実現できる。 また、 D N Aはトラップされたまま、 溶液は流れているの で、 溶液の交換や洗浄にもちいることができる。 狭い部分は、 トラップ する DNAの大きさにより、 0. 0 1 ミクロンから、 5 0ミクロン、 広 い部分は、 狭い部分に比べて断面積で 2倍以上である。 本発明のトラッ プ能力は、 D N Aのサイズにより異なる。 流路径ゃ形状を変えたり、 電 圧や圧力を変えることにより、 トラップできる D NAのサイズを変化さ せることが可能である。 To meet these demands, a simple mechanism formed on the chip is used to selectively capture and hold DNA or similar from liquids containing proteins and various reagents flowing through the flow path (ie, traps). Inexpensive and simple principles that allow DNA to be recovered or DNA to be concentrated, and the trapped DNA to be released by a simple operation and used for the next process Is not yet. Disclosure of the invention 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. Work simultaneously so that they are opposite to each other. The directions of these two forces may be opposite as long as they are opposite to each other. The flow channel 101 has one or more wide portions 105 and one or more narrow portions 106. Although the specific shape of the channel 101 varies as described later, 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. Depending on the size of the DNA to be trapped, 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.
トラップされた DNAは、 電界もしくは圧力のどちらかを強める、 或 いは弱めることにより、 入り口側、 あるいは出口側に放出される。 これ により、 簡単にリ リース、 回収でき、 次の処理に用いることができる。 図面の簡単な説明 第 1図は、 本発明によるトラッピング機構の図であり、 第 2図は、 本 発明を利用する形態の基本の図であり、 第 3図は、 本発明の利用形態の 応用例の図であり、 第 4図は、 本発明のトラッピング機構の構成例の図 であり、 第 5図は、 本発明のトラッピング機構を複数配置して効果をあ げる方法の図であり、 第 6図は、 効果の異なる本発明を直列に接続して 分離を行う方法の図であり、 第 7図は、 本発明を利用して血液等を分析 する構成の図であり、 第 8図は、 本発明を実施するための装置構成例の 図であり、 第 9図は、 実施例における圧力場のみでの D N Aの泳動速度 の図であり、 第 1 0図は、 実施例における電場のみでの D N Aの泳動速 度の図であり、 第 1 1図は、 実施例における圧力場と電場を両方かけた 場合の D N Aのトラッピングの図であり、 第 1 2図は実施例における卜 ラッピングが起こる電圧と圧力の範囲の図であり、 第 1 3図は、 実施例 における逆接続でのトラッピングが起こる電圧と圧力の範囲の図であり . 第 1 4図は、 実施例における圧力場と電場を両方かけた場合の D N Aの 移動距離と時間の関係の図であり、 第 1 5図は、 実施例における圧力場 のみの場合の D N Aの移動距離と時間の関係の図であり、 第 1 6図は、 実施例における電場のみの場合の D N Aの移動距離と時間の関係の図で あり、 第 1 7図は、 現在のトラッビング現象の説明図であり、 第 1 8図 は、 本発明に液体供給装置を付加した応用例の図である。 発明を実施するための最良の形態 The trapped DNA is released to the entrance or exit by increasing or decreasing either the electric field or the pressure. As a result, it can be easily released and collected, and can be used for the next processing. BRIEF DESCRIPTION OF THE FIGURES 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, and 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, and FIG. 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, and 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, and 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, and Fig. 13 is a diagram of the range of voltage and pressure at which trapping occurs in reverse connection in the embodiment. Yes. 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
図 2に本発明に基づき D N Aをトラップ、 リ リースする最も基本的な 形態を示す。 トラップ機構 2 0 1は本発明による、 広い部分と狭い部分 を持った流路である。 入り口 （リザーバ） 2 0 2は D N Aの入り口であ り ここに D N Aが含まれた液体を入れる。 液体は流路 2 0 4、 トラップ 機構 2 0 1を通り、 出口 （ゥエステ） 2 0 3へ流れる。 入り口 2 0 2と 出口 2 0 3のどちらかまたは両方から圧力源につながった管をもちいて 圧力を加える。 また入り口 2 0 2と出口 2 0 3には電極などをもちいて 同時に電界を加える。 圧力により入り口 2 0 2から出口 2 0 3へ、 液を 搬送し、 同時に入り口 2 0 2に正、 出口 2 0 3に負の電圧を加える。 こ れにより入り口 2 0 2の液のうち D N Aだけがトラップ機構 2 0 1 にト ラップされる。 入り口 2 0 2の液が大部分出口 2 0 3に流れたあと、 電 界を強める、或いは圧力を弱めるまたは切る或いは逆転することにより。 入り口 2 0 2側に濃縮された DNAを取り出すことができる。 あるいは 圧力を強める、 または電界を弱めるか切るか反転することにより、 出口 2 0 3側に濃縮された D NAを取り出すことができる。 Figure 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 (ゥ 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. As a result, only the DNA in the liquid at the entrance 202 is trapped by the trap mechanism 201. After the liquid at inlet 202 flows mostly to outlet 203, by increasing the electric field, or reducing or turning off or reversing the pressure. The concentrated DNA can be taken out at the entrance 202 side. Alternatively, by increasing the pressure, or weakening, turning off, or reversing the electric field, the concentrated DNA can be taken out at the outlet 203 side.
図 3の上の図は、 本発明の形態であり、 図 2に加え、 入り口 3 0 1側 に、 濃縮された DN A回収流路 3 0 2と、 電極を加えるリザーバを兼ね る DNA回収口 3 0 3が設けられている。 入り口 3 0 1から DNAを圧 力によって、 トラップ機構 2 0 1へ流し、 DNA回収口 3 0 3に正、 出 口 2 0 3に負の電圧を印加する。 十分トラップされたあと、 圧力を弱め ることにより、 濃縮された D N Aを流路 2 0 4を経由して、 DNA回収 流路 3 0 2、 D NA回収口 3 0 3に回収することができる。 回収路が、 2 0 3出口側にあっても同様に回収できる。  The upper diagram in FIG. 3 shows an embodiment of the present invention. In addition to FIG. 2, 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. After sufficient trapping, by reducing the pressure, 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.
図 3の下図は、 本発明の利用形態であり、 DNA回収流路 3 0 2の先 に、 検出部 3 0 4が取り付けられている。 検出部 3 0 4は P C Rや LA MPチャンパ一や、 D N Aチップが考えられる。 また、 各種電気泳動力 ラムであってもよい。 電気泳動を行う場合は、 電気泳動開始時に、 ブラ グ状にサンプルを開始点につめる必要がある。 通常、 十字路などを用い てこれを実現するが、 本発明によりリ リースされた D NAはプラグ状に 固まってリ リースされるため、 この作業を省略してもよい。 また、 検出 部 3 0 4は、 検出以外の後処理を行う要素に置き換えられる。 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. Also, 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.
図 1 8は、 図 3下段の構成に、 液体供給装置 1 8 0 1 を付加したもので ある。  FIG. 18 shows a configuration obtained by adding a liquid supply device 1801 to the configuration shown in the lower part of FIG.
圧力により入り口 3 0 1から出口 2 0 3へ、 D N Aが含まれた液を搬送 し、 同時に入り口 3 0 1に正、 出口 2 0 3に負の電圧を加える。 これに より入り 口 3 0 1の液のうち D N Aだけがトラップ機構 2 0 1 にトラッ プされる。 十分 D N Aがトラップされた後、 D N Aが圧力流れから受け る力と、 電界から受ける力を保ったまま、 入り口 3 0 1からの液の供給 を中止し、 液体供給装置 1 8 0 1から、 交換液リザ一バ 1 8 0 2に格納 されている交換液を供給することにより、 D N Aをトラップしたまま、 D N Aの周囲の液を交換できる。 また、 交換液を必要な時間連続して流 すことにより、 交換液による D N Aの処理や、 D N Aの洗浄を行うこと ができる。 その後、 電界を強める、 或いは圧力を弱めるまたは切る或い は逆転することにより、 液交換し、 処理あるいは洗浄された D N Aを回 収し、 同様に次の処理に移すことが可能である。 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. As a result, of the liquid at the inlet 301, only the DNA is trapped by the trap mechanism 201. After the DNA has been sufficiently trapped, 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 By supplying the exchange liquid stored in the liquid reservoir 1802, the liquid around the DNA can be exchanged while the DNA is trapped. In addition, 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.
図 4は、 本発明トラップ機構を構成する流路 1 0 1の流路幅の狭い部 分と流路幅の広い部分を実現するさまざまな形態であり、 上段の図は、 単純に幅の異なる流路を連結することに実現される。 幅は、 チップの水 平方向でもよいし、 深さ方向でも、 その両方でもよい。 中段の図は、 流 路の幅を変える代わりに、 流路壁 4 0 1内にビーズなど障害物 4 0 2の 詰め物をすることにより、トラップ機構を構成する。図 4の下段の図は、 多孔質の膜や樹脂等の多孔質物質 4 0 3を流路壁 4 0 1内に形成するこ とによりこれを実現した例である。 光硬化樹脂などを用いることにより 簡単に実現できる。  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. In the middle diagram, instead of changing the width of the flow path, 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.
図 5は、 本トラップ機構を複数並列に接続するための形態であり、 5 0 1は流路入り口、 5 0 2は流路出口であり、 スループッ トやトラップ 量を向上することが可能である。 上段の 2つは、 平面上に複数集積化す る方法の形態であり、 本トラップ機構は非常に微小なため、 集積化によ り 1 0 0 0 0倍程度の能力向上は容易に得られる。 また、 下段は、 板状 のものにあけられた孔とそれに隣接する空間により トラップ機構を実現 するものであり、 さらに高い集積度が簡単に得られる。 Fig. 5 shows a configuration for connecting multiple trap mechanisms in parallel. Reference numeral 01 denotes an inlet of the flow channel, and 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.
図 6は、 トラップできるサイズの異なる トラップ機構 6 0 1 を直列に 接続路 6 0 2を介して、 あるいは介さずに直接、 直列に接続した形態で あり、 これにより異なるサイズの DNAを異なる トラップ機構にトラッ プすることができる。 これにより簡単にサイズによる分離が実現でき、 分析や DNAのフィルタリングに応用できる。 接続路 6 0 1に回収路 6 0 3を接続した場合は、サイズごとに異なる DN Aを分けて回収したり、 後処理を行うことが可能である。  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. When 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.
図 7は、 本機構を用いた D N A分析装置の構成である。 入り 口 7 0 1 に注入された微量な血液、 血清、 大腸菌のような検体を含む液は、 前処 理部 7 0 2により、 酵素の非活性化処理、 アルカリや酵素を用いた細胞 壁やウィルス壁の破砕が行われる、 次に既に述べた様々な形態の本発明 を利用した D N Aトラップ機構 7 0 3に注入され、 破砕された細胞壁や 淡白質、 イオン類から DNAのみを濃縮し、 回収する。 リ リースされた D N Aは検出部 7 0 4により選択的検出される。 検出部 7 04は P C R 法や L AM P法や電気泳動力ラムなどが考えられる。 これにより、 血液 や細胞、 菌類、 或いは、 血清中に薄まったウィルス中の D NAを濃縮し て、 チップ上で、 高感度に検出可能である。  Fig. 7 shows the configuration of a DNA analyzer using this mechanism. The liquid containing a small amount of blood, serum, or E. coli, which is injected into the inlet 701, contains a sample, such as enzyme, which is deactivated by the pretreatment section 70 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. As a result, DNA in blood, cells, fungi, or virus diluted in serum can be concentrated and detected on a chip with high sensitivity.
図 8は、本発明を用いて D NAをトラップした実施例のひとつである。 YOY01で染色した DNA 8 0 1は、 蛍光顕微鏡で 1分子観察が可能 である。 ここでは T 4— D NA (大きさ 1 6 0 k B P) あるいは λ— D NA (大きさ 4 8 k B P) を用いた。 DNAは、 0. 5 T B E緩衝液に、 メルカプトエタノール、 グルコースォキシダーゼ、 力タラ一ゼ、 ダルコ —スと一緒に含まれており、 溶存酸素によって観察中に D NAが切れる のを抑えてある。 また電気浸透流をおさえるために、 ポリビニルピロリ ドンが含まれている。 電源 8 0 2により電圧を印加する。 圧力計 8 0 3 は、 シリンジ 8 0 4を用いて加えた圧力を計測する。 白金線 8 0 5を用 いて入り口と出口に電圧を印加する。 8 0 6はガラス板であり、 8 0 7 はシリコンゴムであり、 両者は入り口と出口を密閉するために用いられ る。 8 0 8は、 石英製のチップであり、 図 1のようなクサビ型が連続し たようなトラップ機構が、 図 2のように配置されている。 クサビの最も 細い部分は 0. 6ミクロンであり、 太い部分は 5ミクロン、 クサビの周 期は 5 0ミクロン、 繰り返し回数は、 8回である。 深さは 0. 5ミクロ ンである。 8 0 9はレンズであり、 D N Aの様子を拡大観察する。 8 1 0はダイクロイツクミラー、 8 1 1はミラ一、 8 1 3は励起光 （4 9 0 n m) であり、 蛍光観察のための光学系である。 DNAからの蛍光 8 1 4を、 高感度 C C Dカメラ 8 1 2によって観察する。 FIG. 8 shows one example of trapping DNA using the present invention. One molecule of DNA801 stained with YOY01 can be observed with a fluorescence microscope It is. Here, T 4-D NA (size 160 k BP) or λ-D NA (size 48 k BP) was used. 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. Also contains polyvinylpyrrolidone to suppress electroosmotic flow. 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. 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.
図 9は、 まず圧力だけをかけて D N Aを泳動させた例であり、 図は圧 力差およそ 4 0 P a以下のものであるが、 速度に差は見られたが、 この ように小さな圧力差であっても、 大きい DNA (T 4 ) も、 小さい DN A (そのフラグメント） もトラップされなかった。 圧力差が大きくなる につれ、 DN Aは益々容易にこの流路を通過した。 これはクサビの方向 を逆にしても同様であった。 これは、 後述される D N Aのトラップの後 のリ リースに利用できる。  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.
図 1 0は、 電場のみをかけて D N Aを泳動した例である。 この図はも つとも電圧が低い （ 0. I V以下） の例であるが、 DNAは簡単にこの 流路を通過した。 より大きい、 電圧差では、 更に容易にこの流路を通過 した。 これはクサビの方向を逆にしても同様であった。 これは、 後述す る卜ラップされた D N Aのリ リースに利用できる。 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.
図 1 1は、 クサビの方向に対して、 電界が D N Aに及ぼす力が、 電界 による力の方向 1 1 0 1になるように電圧を 6 V印加し、 圧力流れによ る力の方向 1 1 0 2に圧力を 5 k P a印加した場合の例である。 このと き、 T 4—DNAは、 図中 D N A 1 0 2で表示された位置にトラップさ れ、 長時間 1 0分ほどたつても動かなかった。 また、 トラップされた D NAは電界を切ると圧力流れによる力の方向 1 1 0 2の方向に直ちに、 リ リースされた。  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 This is an example in which a pressure of 5 kPa is applied to 02. At this time, 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.
図 1 2は、 図 1 1における T 4一 D NAのトラップが起こる電圧と圧 力の条件範囲である。 1 2 0 1はトラップの起こる範囲 （斜線部） であ り、 この範囲の中の条件で、 T 4一 D N Aが確実にトラップされた。 お よそ 3 k P a以上の領域で、 圧力流とつりあう電圧の付近でトラップが 見られる。 トラップが起こる電圧の範囲は、 圧力が増加するにつれ、 広 くなつた。 また、 T 4一 DNAよりも小さい DNAは、 この条件のすこ し内側で、 リ リースされ、 トラップにはサイズ依存性があることが示さ れた。  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.
図 1 3は、 図 1 1において、 圧力と電圧の向きを両方とも反転した場 合のトラップがおきる範囲である。 図 1 2と同様に、 圧力が増加するに つれ、 電圧のトラップ範囲はひろくなつた。  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.
図 1 2及び図 1 3中の実線は、 DN Aに及ぼす、圧力流れからの力と、 電界からの力が、 つりあう条件である。 このことから、 本トラップは、 D N Aに及ぼす力において、 圧力による力と電界による力が、 つりあう 条件の 3 0 %から 1 7 0 %の範囲で起こり、 形状と DNA分子によって 決まるある閾値よりも強い力を印加する必要があることが判る。 The solid lines in 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.
図 1 4は、 図 1 2において、 トラップ領域の下側における、 大、 小 D NAの移動を示したものである。この図から、このような形状において、 圧力と電場を両方逆向きに印加した場合は、 泳動速度に著しいサイズ依 存性がみられた。 トラップ領域の上側においても同様な著しいサイズ依 存性がみられた。 これは、 ゲルやキヤピラリーを用いた電気泳動法に代 わる、 D N Aサイズ分離法である。  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.
図 1 5、 図 1 6は圧力場、 電場のみの場合において、 図 1 4と同様な プロッ トを行ったものであるが、 圧力場、 電場のみの場合はサイズによ る泳動速度の差はほとんどないことが判る。  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.
クサビ型の、 最も狭い部分と、 広い部分の大きさを変えて、 トラップ を行った。 広い部分は、 トラップされている DN Aの大きさからは十分 無限大とおもわれる 1 0 0ミクロンを越えても問題なく トラップできる ことが判った。 狭い部分を変化させると、 同じ圧力、 電場においても、 トラップできる D N Aのサイズが変化し、 0. 6ミクロンでは、 1 0 0 O b p以上の DNAがトラップされ、 0. 3ミクロンでは 5 0 O b p以 上の D NAがトラップされた。 また、 5 0ミクロンのものでは、 D NA のトラップは見られなかった。 この結果より、 小さい DNAをトラップ するには、 最も狭い部分のサイズが小さい方が有利で、 また狭い部分の 大きさや電圧や圧力の大きさを最適化することにより、 特定の大きさ以 上の D N Aをトラップできることがわかる。 また、 D N Aをトラップす るのに有効な狭い部分の幅は、 D N Aの大きさから考えて、 0. 0 1 ミ クロンから 5 0ミクロンの間と考えられる。  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. When the narrow part is changed, 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.
次に第 2の実施例を示す。 第一の実施例と同様に図 8に示した蛍光観 察装置を用いる。 ここで、 泳動チップ 8 0 8は図 1 8に示すように、 入 り口 3 0 1 と出口 2 0 3の他に、 DNA回収流路 3 0 2、 DNA回収口 3 0 3と、 液体供給装置 1 8 0 1、 交換液リザーバ 1 8 0 2を付加した ものを用いる。 トラップ機構 2 0 1は、 第 1の実施例と同様にクサビの 最も細い部分は 0. 6 ミクロンであり、 太い部分は 5ミクロン、 クサビ の周期は 5 0ミクロン、 繰り返し回数は、 8回である。 深さは 0. 5ミ クロンである。 0. 5 T B E緩衝液に、 メルカプトエタノール、 ダルコ 一スォキシダ一ゼ、 カタラーゼ、 グルコースを付与し溶存酸素によって 観察中に D N Aが切れるのを抑え、 また電気浸透流をおさえるために、 ポリビニルピロリ ドンを付与した、 緩衝液を用意する。 これを緩衝液 A とする。 まず流路全体を緩衝液 Aで満たす。 実施例一と同様に調製した D NA溶液に、 表面に C O〇H基を付与したポリスチレンビーズを混入 した溶液を、 入り口 3 0 1に入れる。 交換液リザ一バ 1 8 0 2には、 緩 衝液 Aを入れる。 入り口 3 0 1には、 圧力を印加するシリンジと白金電 極を実施例一と同様に接続する。出口 2 0 3には、白金電極を接続する。 交換液リザーバ 1 8 0 2と、 DNA回収ロ 3 0 3には、 それぞれ、 圧力 を印加するシリンジを接続する。 この 4つの接続は実施例一と同様にシ リコーンゴムでそれぞれ密閉されている。 Next, a second embodiment will be described. As in the first embodiment, the fluorescence observation shown in FIG. Use a monitoring device. Here, as shown in Fig. 18, 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. Use the one with the device 1801, and the replacement fluid reservoir 1802 added. As in the first embodiment, 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. 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 Prepare a buffer solution. This is called Buffer A. First, the entire channel is filled with buffer A. A solution obtained by mixing polystyrene beads having a CO〇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. Fill buffer solution A into the replacement fluid reservoir 1802. 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.
まず、 入り口 3 0 1に 8 k P aの圧縮の圧力を加えると同時に、 出口 2 0 3と入り口 3 0 1の間に 1 0 Vの電圧を、 出口 2 0 3側が負になる ように加える。 この時、 液体供給装置 1 8 0 1、 DNA回収流路には D N Aが流れないように、 交換液リザ一バ 1 8 0 2、 DNA回収ロ 3 0 3 に印加する圧力を調整する。 入り口 3 0 1にいれた、 DNAと溶液とポ リスチレンビーズは、 次々とトラップ機構 2 0 1に流れ込み、 DNAは トラップされ、 濃縮されるが、 ビーズは出口 2 0 3へ流れ去った。 十分 D NAがトラップされた後、 交換液リザ一バへ印加する圧力を増加し、 入り口 3 0 1に印加する圧力を弱めると、 入り口 3 0 1からの DNAと ポリスチレンビーズの供給が止まり、 トラップされた D N Aは交換液リ ザーバからの液体にさらされた。 これにより、 トラップ機構に存在する ポリスチレンビーズは完全に出口 2 0 3へ流れ去り、 DNAは洗浄され た。 次に、 印加している電界と圧力を同時に 0にし、 DNA回収口 3 0 3に引っ張りの圧力を加える。 この時、 入り口 3 0 1 と交換液リザーバ 1 8 0 2から液体が流出しないような引っ張りの圧力を加える。 トラッ プされた D NAは DNA回収流路 3 0 2を通って、 DNA回収口に回収 された。 以上により、 DN Aとポリスチレンビーズの混合物から、 DN Aのみを濃縮して抽出し、 洗浄し、 回収することができた。 First, 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. . At this time, 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. sufficient After the trapping of DNA, increasing the pressure applied to the exchange solution reservoir and decreasing the pressure applied to the inlet 301, the supply of DNA and polystyrene beads from the inlet 301 stops, and trapping occurs. The exposed DNA was exposed to fluid from the exchange fluid reservoir. As a result, the polystyrene beads existing in the trap mechanism completely flowed to the outlet 203, and the DNA was washed. Next, the applied electric field and pressure are simultaneously reduced to 0, and a pulling pressure is applied to the DNA recovery port 303. At this time, a pulling pressure is applied so that the liquid does not flow out from the inlet 301 and the exchange liquid reservoir 1802. The trapped DNA passed through the DNA recovery channel 302 and was recovered at the DNA recovery port. As described above, it was possible to concentrate, extract, wash, and recover only DNA from the mixture of DNA and polystyrene beads.
図 1 7はトラップ力の説明である。 1 7 0 1は電場による力であり、 D N Aが電場から受ける力は壁面からの距離によらず一定である。 これ に対して、 1 7 0 2は圧力流による力であり、 D N Aが圧力流からうけ る力は、 壁面付近が小さく、 中央が大きい。 従って、 最も狭い部分近辺 では、 壁面付近では電場による力が強くなり、 中央部 1 7 0 3では非常 に強い圧力による力ができる。 ここを流れる粒子状のものは、 壁面から すり抜けることが可能であるが、 D N Aのような長い分子は、 すり抜け る過程で、 逃げ出そうとする DNA 1 7 0 4のように長い分子のどこか が中央の流れに引きずられトラップ部に戻される。 これによつて、 長い 分子のみトラップされる。  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. In contrast, 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.
トラップ中の D N Aを詳細に観察すると、 図 1 7で示すように運動し ながら トラップされる DN Aが観察されこのトラップ力の説明が裏付け られる。  When the DNA in the trap is observed in detail, the DNA that is trapped while exercising is observed as shown in Fig. 17, supporting the explanation of the trapping force.
以上に述べたとおり、 本発明による D N Aトラップ機構により、 液体 より D NA、 或いは DNAを含む長い分子のみをトラップし、 必要に応 じてリ リースすることが可能である。 これにより、 チップ上で、 DNA を取り出すための前処理の溶液から DNAのみを回収する機構、 DNA を残して緩衝液や溶液を交換する作業、 あるいは、 非常に薄まった DN Aを濃縮し P C Rや LAMP法、 DN Aチップなどの検出感度を上げた り、 チップ上で扱いやすい液量に調整したりすることが容易になる。 こ れにより、 血液中の白血球や、 ウィルス、 病原体の D N Aの分析や診断 が容易になり、 また、 抗体検出ではなく抗原を検出することにより病気 の診断がより早く、 正確になる効果がある。 また、 トラップ機構や泳動 速度のサイズ依存性を利用した、 新しい、 DNA分離法や、 DNAフィ ル夕リング法、 スクリーニング法も容易に構成できる。 また、 DNAを 利用した蛋白質の合成や、 D NAを用いた分析法、 DNAを用いたデバ イスの開発において、溶液中の DNAのみを一時的に固定できることは、 溶液の交換や、 DNAのマ二ュピレ一シヨン、 D NAの観察を容易にし、 あらゆる波及効果が期待できる。 本発明は、 DNAに特化して説明を行 つたが、 同様の特性をもつ、 RN Aや長鎖線状分子に応用できることは 容易に推測できる。 産業上の利用可能性 As described above, 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. In addition, new DNA separation methods, DNA filtering methods, and screening methods can be easily configured using the trapping mechanism and size dependence of migration speed. In addition, in the synthesis of proteins using DNA, the analysis method using DNA, and the development of devices using DNA, 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. Although 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
以上のように、 本発明にかかる トラップ · リ リース装置は、 介在物の ある溶液から、 D NAあるいは電荷をもつ線状分子を選択的にトラップ し、 簡単な操作でリ リースできる。 このため、 特定の分子を濃縮 · 抽出 したり、 溶媒の交換や、 分子の洗浄に有用であり、 特にチップ上で細胞 から D NAや R NAを取り出す前処理に適している。  As described above, the trap / release device according to the present invention 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.

Claims

請 求 の 範 囲 The scope of the claims
1 . D N A或いは電荷をもつ線状の分子が含まれる液体を流すことがで きる流路において、 当該流路が少なくとも一つ以上の狭い部分と当該狭 い部分に連結された当該狭い部分の断面積の 2倍以上の断面積を有する 広い部分を有し、 当該流路内において当該液体を移動させる圧力差を当 該液体に印加することと、 当該圧力差により当該液体が移動する方向と 反対の方向に当該 D N A或いは電荷をもつ線状の分子を移動させる電界 を当該液体に印加することを特徴とする当該 D N A或いは電荷をもつ線 状の分子のトラップ · リ リース装置。 1. In a flow path through which a liquid containing DNA or charged linear molecules can flow, the flow path has at least one narrow portion and a cut in the narrow portion connected to the narrow portion. Has a wide portion having a cross-sectional area of at least twice the area, applies a pressure difference to move the liquid in the flow path to the liquid, and opposes a direction in which the liquid moves due to the pressure difference. And applying an electric field to said liquid to move said DNA or charged linear molecules in the direction of the trap or release of said DNA or charged linear molecules.
2 . 請求の範囲第 1項記載の流路の狭い部分と広い部分において、 当該 狭い部分の断面積と当該狭い部分に連結された当該広い部分の断面積が 連続的に変化することを特徵とする D N A或いは電荷をもつ線状の分子 のトラップ · リ リース装匱。  2. In the narrow part and the wide part of the flow path according to claim 1, the cross-sectional area of the narrow part and the cross-sectional area of the wide part connected to the narrow part continuously change. The trap or release line of DNA or charged linear molecules.
3 . 請求の範囲第 2項記載の狭い部分において、 その断面の最も狭い箇 所の幅が 0 . 0 1 ミクロンから 5 0ミクロンであることを特徵とする D N A或いは電荷をもつ線状の分子のトラップ . リ リース装置。 3. In the narrow part of claim 2, the width of the narrowest part of the cross section is from 0.01 micron to 50 microns, and the width of the DNA or the linear molecule having electric charge is characterized. Trap. Release device.
4 . 請求の範囲第 1項ないし第 3項記載のトラップ ' リ リース装置が並 列或いは直列に接続されたことを特徵とする D N A或いは電荷をもつ線 状の分子のトラップ · リ リース装置。  4. A trap and release device for a DNA or charged linear molecule, wherein the trap 'release devices according to claims 1 to 3 are connected in parallel or in series.
5 . 請求の範囲第 4項記載の D N A或いは電荷をもつ線状の分子のトラ ップ · リリース装置が、 細胞壁を破砕する前処理部、 ならびに、 P C R 法や L A M P法や電気泳動法を用いた選択的検出部を備えることを特徵 とする分析 · 診断装置。  5. The trap or release device for DNA or charged linear molecules according to claim 4 uses a pretreatment unit for crushing cell walls, and uses a PCR method, a LAMP method, or an electrophoresis method. An analysis / diagnosis device comprising a selective detection unit.
6 . 請求の範囲第 1項において、 トラップ条件を少しはずした、 電圧や 圧力の範囲で、 D N A或いは電荷をもつ線状の分子がサイズに大きく依 存して泳動することを利用した、 当該分子の分離装置。 6. In the first claim, the voltage and An apparatus for the separation of molecules with the use of DNA or charged linear molecules that largely migrate in the pressure range.
7 . D N A或いは電荷をもつ線状の分子が含まれる液体を流すことがで きる流路で、 かつ、 当該流路が少なくとも一つ以上の狭い部分と当該狭 い部分に連結された当該狭い部分の断面積の 2倍以上の断面積を有する 広い部分を有する流路において、 当該流路内において当該液体を移動さ せる圧力差を当該液体に印加することと、 当該圧力差により当該液体が 移動する方向と反対の方向に当該 D N A或いは電荷をもつ線状の分子を 移動させる電界を当該液体に印加することを特徴とする当該 D N A或い は電荷をもつ線状の分子のトラップ · リ リース方法。  7. A flow path through which a liquid containing DNA or charged linear molecules can flow, and the flow path is connected to at least one narrow portion and the narrow portion connected to the narrow portion. In a flow path having a wide portion having a cross-sectional area twice or more the cross-sectional area of the liquid, applying a pressure difference to move the liquid in the flow path to the liquid, and moving the liquid by the pressure difference A method for trapping and releasing said DNA or charged linear molecules, characterized by applying an electric field to said liquid to move said DNA or charged linear molecules in a direction opposite to the direction in which said DNA or charged linear molecules are moved. .
8 . D N A或いは電荷をもつ線状の分子が含まれる液体を流すことがで きる流路において、 当該流路が少なくとも一つ以上の狭い部分と当該狭 い部分に連結された当該狭い部分の断面積の 2倍以上の断面積を有する 広い部分を有し、 かつ、 当該流路に連結する当該 D N A或いは電荷をも つ線状の分子が含まれる液体とは異なる液体を当該流路に供給する液体 供給装置を有し、 当該流路内において当該 D N A或いは電荷をもつ線状 の分子が含まれる液体、 あるいは当該 D N A或いは電荷をもつ線状の分 子が含まれる液体とは異なる液体を移動させる圧力差を当該液体に印加 することと、 当該圧力差により当該液体が移動する方向と反対の方向に 当該 D N A或いは電荷をもつ線状の分子を移動させる電界を当該液体に 印加することを特徴とする当該 D N A或いは電荷をもつ線状の分子のト ラップ · リ リース装置。  8. In a flow path through which a liquid containing DNA or charged linear molecules can flow, the flow path has at least one or more narrow portions and a cut in the narrow portions connected to the narrow portions. Supply a liquid that has a wide portion with a cross-sectional area of at least twice the area and that is different from the liquid that contains the linear molecules having the DNA or charges connected to the flow channel It has a liquid supply device, and moves a liquid in the flow path that contains the DNA or the linear molecule having the charge, or a liquid different from the liquid that contains the DNA or the linear molecule having the charge. Applying a pressure difference to the liquid; and applying an electric field to the liquid to move the DNA or the linear molecule having a charge in a direction opposite to a direction in which the liquid moves due to the pressure difference. To Trapping and releasing device for DNA or charged linear molecules.
9 . 請求の範囲第 7項記載の D N A或いは電荷をもつ線状の分子のトラ ップ · リ リース方法において、 当該 D N A或いは電荷をもつ線状の分子 が含まれる液体に印加する当該圧力差と当該電界により、 当該 D N A或 いは電荷をもつ線状の分子を当該流路内に停留させた後、 当該流路内に 当該 D N A或いは電荷をもつ線状の分子が含まれる液体とは異なる液体 を供給し、 当該 D N A或いは電荷をもつ線状の分子が含まれる液体とは 異なる液体に当該流路内において当該 D N A或いは電荷をもつ線状の分 子が含まれる液体とは異なる液体を移動させる圧力差を印加し、 かつ、 当該圧力差により当該 D N A或いは電荷をもつ線状の分子が含まれる液 体とは異なる液体が移動する方向と反対の方向に当該 D N A或いは電荷 をもつ線状の分子を移動させる電界を当該液体に印加することを特徴と する当該 D N A或いは電荷をもつ線状の分子のトラップ ·リ リース方法。 9. The trap or release method for DNA or charged linear molecules according to claim 7, wherein the pressure difference applied to a liquid containing the DNA or charged linear molecules is different from the pressure difference applied to the liquid containing the DNA or charged linear molecules. Due to the electric field, the DNA or Alternatively, after the charged linear molecules are retained in the flow channel, a liquid different from the liquid containing the DNA or the charged linear molecules is supplied into the flow channel, and the DNA or the DNA is supplied. Applying a pressure difference that moves the DNA or a liquid different from the liquid containing the charged linear molecules to the liquid different from the liquid containing the charged linear molecules, and Due to the pressure difference, an electric field that moves the DNA or the charged linear molecule in a direction opposite to the direction in which the liquid different from the liquid containing the DNA or the charged linear molecule moves is applied to the liquid. A method for trapping and releasing said DNA or charged linear molecules, wherein said method is applied to said DNA.
1 0 . 請求の範囲第 7項記載の D N A或いは電荷をもつ線状の分子のト ラップ · リ リース方法において、 当該流路内に流した当該 D N A或いは 電荷をもつ線状の分子が含まれる液体について、 当該 D N A或いは電荷 をもつ線状の分子が当該流路内に停留する圧力差と電界強度を求め、 当 該 D N A或いは電荷をもつ線状の分子が含まれる液体に対して求めた当 該圧力差と当該電界強度のうち電界強度のみを求めた当該電界強度より 低い値で印加することを特徴とする当該 D N A或いは電荷をもつ線状の 分子のトラップ ' リ リース方法。 10. The method for trapping and releasing DNA or charged linear molecules according to claim 7, wherein the liquid containing the DNA or charged linear molecules contained in the flow channel. The pressure difference and electric field strength at which the DNA or the charged linear molecule stays in the flow path are determined, and the liquid or the liquid containing the DNA or the charged linear molecule is determined. A method for trapping and releasing the DNA or charged linear molecules, wherein the voltage is applied at a value lower than the electric field strength obtained from the pressure difference and the electric field strength.
1 1 . 請求の範囲第 7項記載の D N A或いは電荷をもつ線状の分子のト ラップ . リ リース方法において、 当該流路内に流した当該 D N A或いは 電荷をもつ線状の分子が含まれる液体について、 当該 D N A或いは電荷 をもつ線状の分子が当該流路内に停留する圧力差と電界強度を求め、 当 該 D N A或いは電荷をもつ線状の分子が含まれる液体に対して求めた当 該圧力差と当該電界強度のうち電界強度のみを求めた当該電界強度より 高い値で印加することを特徵とする当該 D N A或いは電荷をもつ線状の 分子のトラップ · リ リース方法。 11. A trap of DNA or a linear molecule having a charge according to claim 7. In the release method, a liquid containing the DNA or the linear molecule having a charge flowing in the flow channel. The pressure difference and electric field strength at which the DNA or the charged linear molecule stays in the flow path are determined, and the liquid or the liquid containing the DNA or the charged linear molecule is determined. A method for trapping and releasing the DNA or charged linear molecules, wherein the applied voltage is applied at a value higher than the electric field strength obtained from the pressure difference and the electric field strength alone.
1 2. 請求の範囲第 7項、 第 9項、 第 1 0項、 第 1 1項に記載の DNA 或いは電荷をもつ線状の分子のトラップ · リ リース方法において、 当該 圧力差が当該 DNAあるいは電荷をもつ線状の分子に及ぼす力と当該電 界強度が当該 D N Aあるいは電荷をもつ線状分子に及ぼす力がつりあう 圧力差と電界強度を共に 0. 3から 1. 7倍した圧力差と電界強度を当 該流路内に流した当該 DN A或いは電荷をもつ線状の分子が含まれる液 体に印加することを特徵とする当該 DNA或いは電荷をもつ線状の分子 のトラップ · リ リース方法。 1 2. The method for trapping and releasing DNA or charged linear molecules according to claim 7, 9, 9, 10 or 11, wherein the pressure difference is equal to the DNA or the linear molecule. The force exerted on the charged linear molecule and the force exerted on the DNA or charged linear molecule by the electric field balance the pressure difference and the electric field obtained by multiplying both the pressure difference and the electric field strength by 0.3 to 1.7. A method for trapping and releasing the DNA or charged linear molecules, characterized in that the strength is applied to a liquid containing the DNA or charged linear molecules flowing through the flow channel. .
1 3. 請求の範囲第 8項に記載の DNA或いは電荷をもつ線状の分子の トラップ · リ リース装置において、 当該圧力差が当該 D N Aあるいは電 荷をもつ線状の分子に及ぼす力と当該電界強度が当該 D N Aあるいは電 荷をもつ線状分子に及ぼす力がつりあう圧力差と電界強度を共に 0. 3 から 1. 7倍した圧力差と電界強度を当該流路内に流した当該 D NA或 いは電荷をもつ線状の分子が含まれる液体に印加することを特徵とする 装置。  1 3. The apparatus for trapping and releasing DNA or charged linear molecules according to claim 8, wherein the force exerted by the pressure difference on the DNA or charged linear molecules and the electric field are applied. The pressure difference and the electric field strength at which the force exerted on the DNA or charged linear molecule is balanced by 0.3 to 1.7 times both the pressure difference and the electric field strength are applied to the DNA or linear molecule. Or an apparatus characterized in that it is applied to a liquid containing charged linear molecules.
PCT/JP2003/003747 2002-03-26 2003-03-26 Dna trap/release apparatus using channel and method of trapping and releasing dna WO2003080829A1 (en)

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