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WO2025023652A1 - Puce double face pour extraction d'acide nucléique pour test de pcr, cartouche la comprenant, et dispositif d'extraction d'acide nucléique l'utilisant - Google Patents

Puce double face pour extraction d'acide nucléique pour test de pcr, cartouche la comprenant, et dispositif d'extraction d'acide nucléique l'utilisant Download PDF

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Publication number
WO2025023652A1
WO2025023652A1 PCT/KR2024/010470 KR2024010470W WO2025023652A1 WO 2025023652 A1 WO2025023652 A1 WO 2025023652A1 KR 2024010470 W KR2024010470 W KR 2024010470W WO 2025023652 A1 WO2025023652 A1 WO 2025023652A1
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WIPO (PCT)
Prior art keywords
chamber
nucleic acid
absorption
washing liquid
absorption chamber
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PCT/KR2024/010470
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English (en)
Korean (ko)
Inventor
박준희
박찬
박기범
서유진
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Genesystem Co Ltd
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Genesystem Co Ltd
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Priority claimed from KR1020240095536A external-priority patent/KR20250015981A/ko
Publication of WO2025023652A1 publication Critical patent/WO2025023652A1/fr
Pending legal-status Critical Current
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers

Definitions

  • the present invention relates to a double-sided chip for nucleic acid extraction for PCR testing, a cartridge containing the same, and a nucleic acid extraction device using the same.
  • the present invention relates to a double-sided chip for nucleic acid extraction for PCR testing, a cartridge containing the same, and a nucleic acid extraction device using the same, which enables rapid and easy extraction of nucleic acids by moving a biological sample within a single chip, effectively removing foreign substances from the biological sample, and easily separating nucleic acids and buffers.
  • PCR Polymerase Chain Reaction
  • PCR is performed repeatedly through the following three steps.
  • the three steps include: 1) a denaturing step in which a sample solution containing double-stranded DNA is heated to a specific temperature, for example, about 95°C, to separate the double-stranded DNA into single-stranded DNA; 2) an annealing step in which an oligonucleotide primer having a sequence complementary to a specific base sequence to be amplified is provided to the sample solution after the denaturing step, and the sample solution is cooled to a specific temperature, for example, 55°C, together with the separated single-stranded DNA, to bind the primer to the specific base sequence of the single-stranded DNA to form a partial DNA-primer complex; and 3) an extension (or amplification) step in which the sample solution is maintained at an active temperature of DNA polymerase, for example, 72°C, after the annealing step, to form double-stranded DNA based on the primer of the partial DNA-primer complex by DNA polymerase.
  • RNA By repeating the above three steps several times, a target nucleic acid having a specific base sequence can be exponentially amplified.
  • a step of reverse transcribing RNA into DNA can be performed in advance in steps 1) to 3).
  • nucleic acids must be extracted from a biological sample.
  • nucleic acid extraction technique there was a method of solubilizing a sample containing cells with SDS or proteinase K, and then denaturing and removing the proteins with phenol to purify the nucleic acids.
  • the phenol extraction method requires many processing steps, so it takes a lot of time, and the nucleic acid extraction efficiency is greatly affected by the experience and skill of the researcher, so there was a problem that reliability was greatly reduced.
  • kits using silica or glass fibers that specifically bind to nucleic acids have been used to solve this problem. Since the silica or glass fibers have a low binding ratio with proteins and cell metabolites, a relatively high concentration of nucleic acids can be obtained.
  • This method has the advantage of being simple compared to the phenol method, but since it uses chaotropic reagents or ethanol that strongly inhibit enzymatic reactions such as polymerase chain reaction (PCR), these substances must be completely removed, and for this reason, the operation is very cumbersome and takes a long time.
  • PCR polymerase chain reaction
  • the conventional nucleic acid extraction method consists of the steps of 1) adding a cell lysis buffer to the cells to lyse the cells; 2) transferring the lysed cells of step 1 to a filter to fix the nucleic acids; 3) washing the filter of step 2; and 4) recovering the nucleic acids from the filter, and thus has the advantage of being able to extract nucleic acids with high reproducibility in a short period of time and steps.
  • a centrifuge is used when transferring the lysed cells to the filter, washing the filter, or recovering the nucleic acids from the filter. The use of such a centrifuge has the effect of shortening the time, but has the problem of low portability and mobility and complicating the nucleic acid extraction process in the field.
  • nucleic acid extraction methods using magnetic beads there are nucleic acid extraction methods using magnetic beads, nucleic acid extraction methods using syringes and filters, nucleic acid extraction methods using direct lysis buffer (DLB), and nucleic acid extraction methods using Trizol, but the nucleic acid extraction method using magnetic beads requires a magnet to fix the nucleic acid to the wall of the tube, and the use of a pump or valve (automated equipment) or multiple tips and pipettes (manual) to remove the solution.
  • the nucleic acid extraction method using a syringe and filter has a problem that the filter is damaged if a certain amount of force is applied during the process of transferring the nucleic acid using the syringe, making it difficult to extract the nucleic acid.
  • the nucleic acid extraction method using direct lysis buffer has a problem that the sensitivity greatly decreases due to dilution because the DLB (Direct lysis buffer) itself may contain PCR inhibitors, so it must be diluted by 1/10.
  • the nucleic acid extraction method using Trizol has a problem that it uses harmful organic solvents such as phenol or chloroform.
  • the present invention aims to provide a double-sided chip for nucleic acid extraction for PCR testing, which effectively removes foreign substances from a biological sample while moving the biological sample within a single chip without using additional equipment, and easily separates nucleic acids and buffers, thereby quickly and easily extracting nucleic acids, a cartridge including the chip, and a nucleic acid extraction device using the chip.
  • a cartridge including a nucleic acid extraction chip comprises a plate-shaped body part including an absorption chamber that absorbs and removes an inhibitor that inhibits PCR performance from a sample; a front cover coupled to one side of the body part; and a rear cover coupled to the front cover with the body part interposed therebetween; characterized in that nucleic acids are separated from the sample as the sample passes through the absorption chamber.
  • the body part includes an injection chamber into which a biological sample to be examined is injected; a washing solution storage chamber which receives a washing solution for washing an absorbent filled in the absorption chamber; and a waste collection and nucleic acid separation chamber which collects the washing solution that has washed the absorption chamber, passes it through the absorption chamber, and separates and discharges nucleic acids from which inhibitors have been removed, and it is preferable that the sample passes through the injection chamber, the washing solution storage chamber, the absorption chamber, and the waste collection and nucleic acid separation chamber in that order.
  • washing liquid storage chamber the absorption chamber, the waste collection and nucleic acid separation chamber is formed on one surface of the body portion having a predetermined thickness, and the remaining parts of the washing liquid storage chamber, the absorption chamber, the waste collection and nucleic acid separation chamber that are not formed on the one surface are formed on the other surface opposite the one surface.
  • the body part be provided with a nucleic acid chamber in which the nucleic acid separated through the waste collection and nucleic acid separation chamber is stored.
  • the body part includes an injection chamber having an injection port into which a biological sample to be tested is injected, the body part includes a cap that opens and closes the injection port by sliding, and the cap includes a guide projection that is inserted into and guided by a sliding groove formed in the body part, and a sealing member that seals the edge of the injection port at a position where the injection port is closed.
  • the absorption chamber includes a first absorption chamber, a second absorption chamber, and a third absorption chamber that are connected in the vertical direction
  • the first, second, and third absorption chambers include a resin to which the inhibitor is adsorbed, and a mesh-structured barrier film is provided between the first absorption chamber and the second absorption chamber, and between the second absorption chamber and the third absorption chamber, so that the sample passes through the first, second, and third absorption chambers sequentially, and the resin is preferably prevented from moving by the barrier film.
  • the body part is provided with a first tube passage connecting the injection chamber and the washing liquid storage chamber; a second tube passage connecting the washing liquid storage chamber and the absorption chamber; and a third tube passage connecting the absorption chamber and the sink waste collection and nucleic acid separation chamber; and includes a valve that moves up and down with respect to the body part to simultaneously open or close the first tube passage, the second tube passage, and the third tube passage, and it is preferable that the rear cover includes a valve exposure hole that exposes the valve so as to transmit a pressurizing force causing the valve to move upward.
  • an air injection hole is formed in the injection chamber through which air is injected by an air pump, and the sample is pushed and moved toward the absorption chamber by the air pressure provided through the air injection hole, and a docking part connected to the air pump is provided in the body part.
  • a nucleic acid extraction device using a cartridge is characterized by including a plate-shaped body part including an absorption chamber for absorbing and removing an inhibitor that inhibits PCR performance from a sample; a front cover coupled to one side of the body part; and a rear cover coupled to the front cover with the body part therebetween; a cartridge for separating nucleic acid from a sample while the sample passes through the absorption chamber; and a mounting part including an insertion part into which the cartridge is inserted and mounted, and a door provided on the insertion part for opening and closing the insertion part.
  • the body part includes an injection chamber into which a biological sample to be examined is injected; a washing solution storage chamber which receives a washing solution for washing an absorbent filled in the absorption chamber; and a waste collection and nucleic acid separation chamber which collects the washing solution that has washed the absorption chamber, passes it through the absorption chamber, and separates and discharges nucleic acids from which inhibitors have been removed, and it is preferable that the sample passes through the injection chamber, the washing solution storage chamber, the absorption chamber, and the waste collection and nucleic acid separation chamber in that order.
  • At least one of the washing liquid storage chamber, the absorption chamber, the waste collection and nucleic acid separation chamber is formed on one surface of the body portion having a predetermined thickness, and it is preferable that the remaining parts of the washing liquid storage chamber, the absorption chamber, the waste collection and nucleic acid separation chamber that are not formed on the one surface are formed on the other surface opposite the one surface.
  • the body part is provided with a nucleic acid chamber in which the nucleic acid separated through the waste collection and nucleic acid separation chamber is stored.
  • the body part includes an injection chamber having an injection port into which a biological sample to be tested is injected, the body part includes a gap for opening and closing the injection port by sliding, and the cap includes a guide projection that is inserted into and guided by a sliding groove formed in the body part, and a sealing member that seals the edge of the injection port at a position where the injection port is closed.
  • the body part is provided with a first tube channel connecting the injection chamber and the washing liquid storage chamber; a second tube channel connecting the washing liquid storage chamber and the absorption chamber; and a third tube channel connecting the absorption chamber and the sink waste collection and nucleic acid separation chamber; and includes a valve that moves up and down with respect to the body part to simultaneously open or close the first tube channel, the second tube channel, and the third tube channel, and the mounting part is provided with a pressure transmission means for transmitting a pressure to move the valve upward, and the rear cover preferably includes a valve exposure hole exposing the valve so that the pressure transmission means pressurizes the valve.
  • an air injection hole is formed in the injection chamber into which air is injected by an air pump, and the sample is pushed and moved toward the absorption chamber by air pressure provided through the air injection hole.
  • a docking part connected to the air pump is provided in the body part, and a pump connection part inserted into the docking part is provided to which the air pump is connected to the mounting part.
  • a double-sided chip for nucleic acid extraction for PCR testing is characterized by including: an input chamber into which a biological sample to be tested is input; an absorption chamber containing an absorbent that absorbs and removes an inhibitor that inhibits PCR performance from the sample; a nucleic acid chamber containing nucleic acids from which inhibitors have been removed by passing through the absorption chamber; and a body part including the input chamber, the absorption chamber, and the nucleic acid chamber together so that the sample sequentially passes through the input chamber, the absorption chamber, and the nucleic acid chamber.
  • a double-sided chip for nucleic acid extraction for PCR testing comprises: a plate-shaped body having a predetermined thickness; an absorption chamber formed on one surface of the body or the other surface opposite the one surface, and containing an absorbent that absorbs and removes an inhibitor that inhibits PCR performance from the sample; a waste collection and nucleic acid separation chamber formed on one surface of the body or the other surface opposite the one surface, and capturing a washing liquid that washes the absorption chamber, and passing through the absorption chamber to separate and discharge nucleic acids from which inhibitors have been removed.
  • a double-sided chip for nucleic acid extraction for PCR testing according to an embodiment of the present invention, a cartridge including the same, and a nucleic acid extraction device using the same provide the effect of effectively removing foreign substances from a biological sample, easily separating nucleic acids and buffers, and quickly and easily extracting nucleic acids while moving the biological sample within a single chip without using additional equipment.
  • the present invention simplifies the nucleic acid separation procedure by easily separating nucleic acids on a single chip without using additional equipment such as a centrifuge for nucleic acid separation, and improves user convenience by making it easy to carry and move to the field.
  • Figure 1 is a perspective view of a double-sided chip for nucleic acid extraction according to one embodiment of the present invention.
  • Figure 2 is a cross-sectional view of the main part of Figure 1.
  • Figure 3 is a perspective view of Figure 1 from a different angle.
  • Figure 4 is a perspective view of the opposite side of Figure 1.
  • Figure 5 is a drawing showing the path along which the sample flows sequentially in the double-sided chip, indicated by numbers.
  • Figure 6 is a drawing showing the chambers through which the sample passes in sequence.
  • Figure 7 is a plan view of Figure 4.
  • Figure 8 is a plan view of Figure 3.
  • Figure 9 is a perspective view of Figure 1 from a different angle.
  • Figure 10 is a drawing showing the nucleic acid extraction in a nucleic acid chamber.
  • Figure 11 is a drawing showing a cartridge including a double-sided chip
  • Figure 12 is an exploded perspective view of Figure 11.
  • Figure 13 is a drawing showing a state in which a double-sided chip is attached to a rear cover.
  • Figure 14 is a drawing showing a state where the valve closes the tubular path.
  • Figure 15 is a drawing showing a state where the valve has opened the tubular path.
  • Figure 16 is a drawing showing the valve rising by a pressure transmitting means.
  • Figure 17 is a drawing showing the cap sliding to open and close the injection chamber and the bottom surface of the cap.
  • Figure 18 is a perspective view of the cap.
  • Fig. 19 is a front view of Fig. 19.
  • Figure 20 is a drawing showing a cartridge mounted on a nucleic acid extraction device.
  • Figure 21 is a drawing showing how a pump connection part is connected to a cartridge.
  • the present invention relates to a double-sided chip for extracting nucleic acids for PCR tests, and relates to a chip and device for removing an inhibitor that interferes with PCR from a biological sample and separating and obtaining nucleic acids in order to perform PCR on the sample.
  • the double-sided chip for extracting nucleic acids according to the present invention can be utilized not only in cases where extraction of nucleic acids is required for the purpose of diagnosing, treating, or preventing diseases, but also in various fields such as new drug development and detection of environmental hormones where extraction of nucleic acids from samples is required.
  • PCR polymerase chain reaction
  • PCR may use a reaction mixture containing a primer (forward primer, reverse primer), which is an oligonucleotide that can specifically hybridize to a target nucleic acid, a deoxynucleotide triphosphate mixture (dNTP mixture), and a divalent ion such as Mg2+.
  • a primer forward primer, reverse primer
  • dNTP mixture deoxynucleotide triphosphate mixture
  • Mg2+ divalent ion
  • a 'primer' is used to initiate a PCR reaction, and refers to an oligonucleotide or polynucleotide that hybridizes complementarily to template DNA.
  • a primer for a PCR reaction may be a pair of a forward primer (or sense primer) selected from a sense strand identical to the direction of progression of the genetic code of a nucleic acid molecule to be amplified, and a reverse primer (or antisense primer) selected from an antisense strand complementary to the sense strand.
  • the 'sample' refers to genetic material such as nucleic acid that is the target to be amplified or a biological solution containing such genetic material.
  • the 'reaction reagent' is for detecting the target genetic material and may include fluorescent dyes, primers, etc.
  • the primer may be composed of a pair of primers of 15 to 30 bp in length that can bind to both ends of a specific region of the target gene.
  • the DNA polymerase uses an enzyme that does not lose activity even at high temperatures of 90°C or higher.
  • a double-sided chip (1000) for nucleic acid extraction which forms a path and a chamber on both sides of a body part (10) through which a biological sample flows, so that an inhibitory substance (hereinafter, referred to as an inhibitor) that inhibits PCR is adsorbed and removed while passing through the path and chamber, and only nucleic acids are collected and separated into a nucleic acid chamber (300).
  • an inhibitory substance hereinafter, referred to as an inhibitor
  • the body part (10) is formed in a plate shape having a predetermined thickness, and an injection chamber (100), an absorption chamber (200), a nucleic acid chamber (300), a washing solution storage chamber (400), and a weight collection and nucleic acid separation chamber (500) to be described later are provided in the body part (10).
  • a biological sample to be tested is introduced into the injection chamber (100). Before dispensing the sample into the injection chamber, the sample is pretreated so that nucleic acids can be separated. The pretreatment is performed by injecting a predetermined dissolution buffer, for example, a lysis buffer, into the biological sample to destroy the cell wall and cause the nucleic acids to leak.
  • a predetermined dissolution buffer for example, a lysis buffer
  • the lysis buffer is a dissolution buffer used to destroy the cell wall.
  • the sample to which the nucleic acids have been exposed is introduced into the injection chamber (100).
  • the injection chamber (100) is formed on one side of the body part (10) and forms a space in the vertical direction, and is formed so that the width becomes narrower as it goes downward.
  • An injection port (101) into which the sample is injected is provided on the upper side of the injection chamber (100), and an outlet port (102) through which the sample is discharged is provided on the lower side.
  • an inclined guide portion (103) is formed to guide the sample to gather toward the outlet port (102).
  • the sample injected into the injection chamber (100) moves to the washing liquid storage chamber (400) through the discharge port (102) formed at the bottom of the injection chamber (100) by air pressure injected from the top.
  • the air is provided from a pump connected to the bottom of the body part (10), and the air provided by the pump is supplied to the upper side wall of the injection chamber (100).
  • An air injection hole (104) into which air is injected by the pump is formed in the injection chamber (100).
  • the sample can be pushed and moved toward the absorption chamber (200) by the air pressure provided to the air injection hole (104).
  • the absorption chamber (200) is provided to absorb and remove inhibitors that inhibit PCR from the sample.
  • the inhibitors include salts, proteins, and other intracellular chemicals contained in the sample, and act as factors that interfere with the amplification of nucleic acids by the substances when performing PCR, and are removed by the absorbent contained in the absorption chamber (200). Since the inhibitors contained in the sample are absorbed and removed by the absorbent, and only the nucleic acids can be discharged and collected downward, the nucleic acids can be separated simply and quickly.
  • the present invention provides a new concept of a separation device that separates nucleic acids without going through the process of fixing nucleic acids using a predetermined adsorption filter or the like to separate nucleic acids from a conventional sample and then re-separating the nucleic acids from the adsorption filter.
  • the absorption chamber (200) includes first, second, and third absorption chambers (210, 220, and 230).
  • the first, second, and third absorption chambers (210, 220, and 230) are filled with spherical resin particles for absorbing an inhibitor.
  • the spherical resin particles absorb the inhibitor.
  • the first, second, and third absorption chambers (210, 220, and 230) are arranged in a vertical direction, and the sample passes through the first, second, and third absorption chambers (210, 220, and 230) sequentially.
  • a mesh-structured barrier is formed at each boundary of the first, second, and third absorption chambers (210, 220, 230) to prevent the resin contained in one chamber from moving to another chamber.
  • a fastening groove (203) into which the barrier is inserted is formed between the first absorption chamber (210) and the second absorption chamber (220) and between the second absorption chamber (220) and the third absorption chamber (230).
  • a fastening groove (203) is also formed between the absorption chamber inlet (201) and the first absorption chamber (210) and between the third absorption chamber (230) and the absorption chamber outlet (202), so that a barrier is installed to prevent the resin from leaking.
  • the above first, second, and third absorption chambers may be selected to include any one of a cathodic anion exchange resin, a positive cation exchange resin, and a chelate resin.
  • the chelate resin has a weak anode and has a property of adsorbing (capturing) metal divalent ions.
  • the anion exchange resin, chelate resin, and cation exchange resin are formed by including a spherical resin, and the inhibitor is adsorbed onto the spherical resin.
  • the diameter of the spherical resin may be formed to be approximately 0.18 mm to 0.3 mm.
  • the above absorption chamber (200) has three chambers, each of which has a polarity, so that an inhibitor having a polarity in the biological sample can be absorbed and removed in the first, second, and third absorption chambers (210, 220, and 230).
  • the first absorption chamber (210) includes an anion exchange resin
  • the second absorption chamber (220) includes a chelate resin
  • the third absorption chamber (230) includes a cation exchange resin.
  • the first, second, and third absorption chambers (210, 220, and 230) are arranged sequentially from top to bottom, so that the sample passes through the anion exchange resin, the chelate resin, and the cation exchange resin sequentially.
  • the volumes of the anion exchange resin, the chelate resin, and the cation exchange resin can be prepared substantially as 1:1:1.
  • the volume ratio of the anion exchange resin, the chelate resin, and the cation exchange resin is not limited to 1:1:1.
  • the substances included in the biological sample are polar, so that they can be absorbed in each layer while passing through the first, second, and third absorption chambers (210, 220, and 230), and only the nucleic acid can flow downward.
  • the anion exchange resin can include at least one of TMA (Trimethylamine), DMEA (Dimethylethanolamine), and Tertiary Amine
  • the chelate resin can include at least one of a carboxyl group (-COOH), an iminodiacetate acid chelate, and the like.
  • the cation exchange resin can include at least one of a sulfate group (-SO3H), a carboxyl group (-COOH), and a sulfonic acid group (-SO3H).
  • An absorption chamber outlet (202) is formed at the lower side of the third absorption chamber, and an inhibitor is removed from the sample through the absorption chamber outlet (202) and the remaining nucleic acid is discharged downward.
  • the sample moves inside the chip at a speed of 15 ⁇ l/sec or less, and as a result, the flow rate of the nucleic acid discharged through the absorption chamber outlet (202) becomes 15 ⁇ l per second or less.
  • the flow rate per unit time of the nucleic acid discharged through the absorption chamber outlet (202) can be controlled by appropriately forming the cross-sectional area of the absorption chamber outlet (202) through which the nucleic acid passes.
  • the discharge amount of the nucleic acid may increase.
  • the speed at which the sample passes through the absorption chamber (200) relatively increases, it may not be possible to secure enough time for the inhibitor to be sufficiently adsorbed in the first, second, and third absorption chambers (210, 220, 230). Therefore, it was confirmed that the inhibitor could be sufficiently removed when the discharge flow rate of the nucleic acid was adjusted to 15 ⁇ l per second or less.
  • the nucleic acid chamber (300) receives nucleic acids from which inhibitors have been removed by passing through the absorption chamber (200). As illustrated in FIGS. 1 and 2, according to the present embodiment, the nucleic acid chamber (300) has a space whose width narrows from the top to the bottom. As illustrated in FIG. 10, a nucleic acid chamber inlet (310) through which nucleic acids are introduced is formed in the nucleic acid chamber (300). The nucleic acids collected in the nucleic acid chamber (300) can be extracted by a device such as a pipette (330) through an extraction port (320) formed opposite the nucleic acid chamber inlet (310).
  • a device such as a pipette (330) through an extraction port (320) formed opposite the nucleic acid chamber inlet (310).
  • the sample passes through the injection chamber (100), the absorption chamber (200), and the nucleic acid chamber (300) sequentially, and the body part (10) is provided with the injection chamber (100), the absorption chamber (200), and the nucleic acid chamber (300), so that the process of separating and storing nucleic acids from the sample is possible in one body part (10).
  • the washing liquid storage chamber (400) is a chamber provided between the injection chamber (100) and the absorption chamber (200), and accommodates a washing liquid for washing an absorbent filled in the absorption chamber (200).
  • the absorption chamber (200) is filled with a polar resin, and each chamber is filled with a preservation buffer to maintain the polarity of the first, second, and third absorption chambers (210, 220, 230) forming the absorption chamber (200).
  • the washing liquid is provided to wash the preservation buffer.
  • the washing liquid storage chamber (400) is formed on one side of the body part (10) and is formed on the same side as the side on which the absorption chamber (200) is formed.
  • the washing liquid stored in the washing liquid storage chamber (400) moves toward the absorption chamber (200) by the movement pressure of the sample and air, and the absorbent of the absorption chamber (200) is washed first.
  • the washing liquid storage chamber (400) includes a plurality of washing liquid flow paths (440) arranged in parallel in one direction so that the washing liquid can flow.
  • the washing liquid flows into the washing liquid storage chamber inlet (450) on the upper side, passes through the washing liquid flow path (440), and then moves to the absorption chamber (200) through the washing liquid storage chamber outlet (460).
  • the washing liquid flows in a zigzag pattern from upper to lower along the washing liquid flow path (440).
  • the washing liquid flow path (440) includes a first path (401), a second path (402), and a third path (403) having different lengths.
  • the washing liquid storage chamber (440) includes a first storage section (410) formed of a plurality of first channels (401) having a first length to store the washing liquid, a second storage section (420) formed of a plurality of second channels (402) shorter than the first length to store the washing liquid and provided below the first storage section (410), and a third storage section (430) formed of a plurality of second channels (403) shorter than the second length to store the washing liquid and provided below the second storage section (430).
  • the first, second, and third storage sections (410, 420, 430) are sequentially provided from the top to the bottom.
  • the above first, second, and third storage units (410, 420, 430) are formed with a plurality of horizontal flow paths (401, 402, 403) in a zigzag manner, so that sufficient washing liquid can be stored.
  • Waste collection and nucleic acid separation chamber (500) 4. Waste collection and nucleic acid separation chamber (500)
  • the waste collection and nucleic acid separation chamber (500) is provided to collect the washing liquid discharged from the washing liquid storage chamber (400) at the rear end of the absorption chamber (200) and to separate and discharge the nucleic acid.
  • the nucleic acid separated by the waste collection and nucleic acid separation chamber (500) is collected in the nucleic acid chamber (300).
  • the waste collection and nucleic acid separation chamber (500) may be formed on one or the other surface of the body portion (10).
  • At least one of the absorption chamber (200), the washing liquid storage chamber (400), and the waste collection and nucleic acid separation chamber (500) may be formed on at least one of one surface of the body part (10) having a predetermined thickness and the other surface opposite thereto. That is, at least one of the absorption chamber (200), the washing liquid storage chamber (400), and the waste collection and nucleic acid separation chamber (500) is formed on one surface of the body part (10) having a predetermined thickness, and the remainder of the absorption chamber (200), the washing liquid storage chamber (400), and the waste collection and nucleic acid separation chamber (500) that are not formed on the one surface may be formed on the other surface opposite thereto.
  • the washing liquid storage chamber (400) and the absorption chamber (200) are formed on one surface of the body part (10), and the waste collection and nucleic acid separation chamber (500) is formed on the other surface of the body part (10).
  • the absorption chamber (200) may be formed on one side or the other side of the body part (10), and the waste collection and nucleic acid separation chamber (500) may also be formed on one side or the other side of the body part (10). That is, in one embodiment, the absorption chamber (200) and the waste collection and nucleic acid separation chamber (500) may be formed on the same side of the body part (10).
  • the waste collection and nucleic acid separation chamber (500) is provided to collect waste and separate nucleic acids by having the washing liquid passing through the absorption chamber (200) fill the waste chamber (510) and subsequently moving the nucleic acids flowing through the inflow path (570) through the nucleic acid separation path (530).
  • the above waste collection and nucleic acid separation chamber (500) includes an inlet passage (570), a washing liquid collection passage (520), a nucleic acid separation passage (530), and a waste chamber (510).
  • the above inflow path (570) extends from the upper side to the lower side and is a path through which washing liquid and nucleic acid flow. According to the present embodiment, the washing liquid first flows into the inflow path (570), and the nucleic acid flows in following the washing liquid.
  • the inflow path (570) includes a curved path (560) having a predetermined curvature at its end.
  • the washing liquid collection channel (520) is connected to extend downward from the end of the inflow channel (570) so that the washing liquid can flow.
  • the washing liquid collection channel (520) is connected to a curved channel (560) provided at the end of the inflow channel (570) and extends downward.
  • the nucleic acid separation channel (530) is extended in a different direction from the washing solution collection channel (520) at the end of the inflow channel (570), thereby forming a channel through which the nucleic acid flows.
  • the nucleic acid separation channel (530) is extended upward in the opposite direction of the washing solution collection channel (520) while being connected to the curved channel (560).
  • the nucleic acid separation channel (530) is provided with a resistance protrusion (590) protruding from the inner surface.
  • the resistance protrusion (590) has the function of inducing the washing solution to move toward the washing solution collection channel (520) downward when the washing solution is initially introduced.
  • a resistance portion (580) that generates resistance when the washing liquid flows is formed on the round inner surface where the inflow path (570) and the nucleic acid separation path (530) are connected to each other, that is, on the inner surface of the curved path (560).
  • the resistance portion (580) may be formed as a protrusion that protrudes like a sawtooth shape on the inner surface that is satisfied from the end of the curved path (560) toward the nucleic acid separation path (530). The protrusions may be formed at a predetermined interval.
  • the washing liquid flows toward the nucleic acid separation path (530) that extends downward by gravity, and at this time, the washing liquid collides with the resistance portion (580) while passing through the curved path (560), so that the washing liquid can be more easily guided and flow toward the lower waste chamber (510).
  • the width (W2) of the washing solution collection channel (520) is formed wider than the width (W1) of the nucleic acid separation channel (530).
  • the washing solution flows into the inflow channel (570), passes through the curved channel (560), and can flow more smoothly toward the wider washing solution collection channel (520) at the end of the curved channel (560).
  • a washing solution induction channel (524) is formed in the washing solution collection channel (520) that has a narrower width than the width of the washing solution collection channel (520) and is sunken deeper than the bottom of the washing solution collection channel (520).
  • the above washing liquid induction path (524) allows the washing liquid to easily flow downward from the end of the curved path (560) toward the waste chamber (510) by capillary phenomenon.
  • the above-described waste chamber (510) is provided at an end of the washing liquid collection path (520) to provide a space in which the washing liquid is filled.
  • the washing liquid flows in from the bottom of the waste chamber (510) and fills the space of the waste chamber (510).
  • a support rib (501) that protrudes vertically from the bottom surface is formed on the inside of the waste chamber (510).
  • a plurality of the support ribs (501) are provided.
  • one side and the other side of the body part (10) are sealed by adhering a film.
  • the support ribs (501) prevent the surface of the film from sinking into the space of the waste chamber (510) when sealing the other side of the body part (10) with the film.
  • the support rib (501) ensures that the space of the waste chamber (510) can secure a space in which the washing liquid can be stored.
  • a pressure-providing portion (540) is provided on the upper side of the waste chamber (510) so that the washing liquid is filled and the nucleic acid is no longer introduced into the waste chamber (510) by the pressure of the washing liquid.
  • the pressure-providing portion (540) includes a microchannel (550) formed on the upper side of the waste chamber (510). The width of the microchannel (550) is narrower than that of the inflow channel (570), the curved channel (560), the waste collection channel (520), and the nucleic acid separation channel (530). A plurality of micro-spaces (541) are included on the microchannel (550).
  • the internal pressure of the waste chamber (510) increases by the microchannel (550) and the microspace (541). Some of the washing liquid may penetrate into the microchannel (550) and the microspace (541).
  • the microspace (541) is provided in case sufficient pressure is not applied to a portion of the microchannel (550) connected to the waste chamber (510), and even if the washing liquid penetrates through the microchannel (550) near the waste chamber (510), pressure is applied again by the remaining microchannel (550) and the microspace (541).
  • microchannel (550) and microspace (541) prevent the pressure of the waste chamber (510) from increasing rapidly.
  • the pressure providing unit (540) increases by the pressure providing unit (540), the washing liquid is no longer transported toward the waste chamber (510), so that the nucleic acid subsequently introduced through the inflow channel (570) can flow upward through the nucleic acid separation channel (530) due to the repulsive force.
  • the washing liquid that washes away the absorbent of the absorption chamber (200) is collected and separated in the waste chamber (510), and only the nucleic acid can move to the nucleic acid chamber (300) through the nucleic acid separation channel (530).
  • the double-sided chip (1000) may be manufactured and provided in the form of a cartridge (2000).
  • a cartridge (2000) includes a body portion (10), and a front cover (2100) and a rear cover (2200) that are respectively coupled to one side and the other side of the body portion (10).
  • the body portion (100) is positioned between the front cover (2100) and the rear cover (2200), and separates nucleic acids from a sample as the sample passes through the absorption chamber (200).
  • the above body part (10) is formed in a plate shape with a predetermined thickness and includes an injection chamber (100), an absorption chamber (200), a nucleic acid chamber (300), a washing liquid storage chamber (400), and a waste collection and nucleic acid separation chamber (500).
  • an injection chamber 100
  • an absorption chamber 200
  • a nucleic acid chamber 300
  • a washing liquid storage chamber 400
  • a waste collection and nucleic acid separation chamber 500.
  • the body part (10) further includes a tubular flow path (700) in addition to the flow path formed on the surface of the body part (10) so that a sample can be transferred from the injection chamber (100) to the nucleic acid chamber (300).
  • a valve (600) for opening and closing the tubular flow path (700) is provided.
  • the tubular flow path (700) is connected by having both ends inserted into the tube flow path connecting portion (13) provided in the body part (10).
  • the above-described tube-shaped path (700) includes a first tube path provided between the injection chamber (100) and the washing liquid storage chamber (400), a second tube path provided between the washing liquid storage chamber (400) and the absorption chamber (200), and a third tube path provided between the absorption chamber (200) and the waste waste collection and nucleic acid separation chamber (500).
  • the valve (600) simultaneously opens or closes the first, second, and third tube paths.
  • the valve (600) is coupled to the body part (10) so as to be movable in the vertical direction.
  • the pressure rib (610) formed in the valve (600) presses the tubular flow path (700) to maintain the flow path in a closed state. That is, the tubular flow path (700) is in a state where the valve (600) is pressed in the first position as shown in FIGS. 14(a) and 14(b).
  • the pressure rib (610) pressing the tubular flow path (700) moves upward, opening the tubular flow path (700) so that the sample can flow in one direction.
  • a groove is formed in a rear cover (2200) coupled to one side of the body portion (10), and a valve exposure hole (2210) is formed on an upper side of the groove to expose the valve (600) so as to transmit a pressing force to move the valve upward.
  • a pressing force transmission means (3600) provided in the nucleic acid extraction device (3000) transmits a force to move the valve (600) upward through the valve exposure hole (2210). As the valve (600) moves upward by the pressing force, the tubular flow path (700) is opened.
  • a cap (2300) for opening and closing the injection port (101) of the injection chamber (100) is coupled to the upper side of the body part (10). As illustrated in FIG. 17, the gap (2300) is slidably coupled to the upper surface of the body part (10).
  • the cap (2300) includes a guide projection (2370) that is inserted into and guided by a sliding groove (14) formed in the body part (10), and a sealing member (2330) that seals the edge of the injection port (101) at a position where the injection port (101) is closed.
  • the cap (2300) is formed with a cut groove (2310) that faces each other and extends in the longitudinal direction.
  • a catch (2320) is formed protrudingly in the cut groove (2310).
  • a protrusion (15) is formed in the width direction on the upper surface of the body (10), and when the cap (2300) is slid to close the inlet (101), the cut groove (2310) is elastically deformed so that the catch (2320) can pass over the protrusion (15).
  • a sealing member (2330) such as an O-ring that is coupled to the lower surface of the cap (2300) is placed on the edge of the inlet (101) to seal the inlet (101), and the protrusion (15) is maintained in a state of being caught by the catch (2320) so that the cap (2300) closes the inlet (101).
  • the cap (2300) includes a handle (2340), a guide piece (2360) on which the guide projection (2370) is formed, and a position control projection (2350) that regulates the sliding range of the cap (2300).
  • the guide piece (2360) extends downward from both ends of the cap (2300) and is coupled to the upper side of the body part (10), and the guide projection (2370) formed on the inner side of the guide piece (2360) is inserted into the sliding groove (14) of the body part (10).
  • the position control projection (2350) is formed in the longitudinal direction on the upper surface of the cap (2300) so that its end can come into contact with the front cover (2100) or the rear cover (2200) to limit the movement range of the cap (2300).
  • a nucleic acid extraction device (3000) that receives the cartridge and extracts nucleic acids is proposed.
  • the extraction device (3000) includes a cartridge (2000) including the double-sided chip described above, and a mounting portion (3100) on which the cartridge (2000) is mounted. Since the configuration of the cartridge (2000) has been described above, a repetitive description will be omitted.
  • the above-mentioned mounting portion (3100) may include an insertion portion (3200) into which the cartridge (2000) is inserted and mounted, a door (3300) for opening and closing the insertion portion (3200), a select bar (3400) for controlling the nucleic acid extraction device (3000), and a display portion (3500) for externally displaying whether the nucleic acid extraction device (3000) is operating.
  • the insertion portion (3200) is provided so that two cartridges (2000) can be inserted.
  • the number of cartridges (2000) that can be accommodated is not limited thereto.
  • the above select bar (3400) can be implemented to enable selection of the on/off operation and operation time of the nucleic acid extraction device (3000).
  • the display unit (3500) is provided on the upper surface of the mounting unit (3100) to visually check the operation progress status of the nucleic acid extraction device (3000).
  • the select bar (3400) is provided in a knob type, and the nucleic acid extraction device (3000) programmed under predetermined conditions can be operated by rotating the select bar (3400).
  • the mounting portion (3100) is provided with a pump connection portion (3700) for supplying air to the cartridge (2000).
  • a docking portion (11) for receiving air pressure from an air pump is provided in the body portion (10) of the cartridge (2000), and a docking hole (2230) is provided to expose the docking portion (11) at the lower side when the front cover (2100) and the rear cover (2200) are coupled to each other.
  • the upper portion of the pump connection portion (3700) is inserted into the docking portion (11), and a pump is connected to the lower portion.
  • the present invention relates to a double-sided chip (1000), a cartridge (2000), and a nucleic acid extraction device (3000) for extracting nucleic acids.
  • the present invention improves the nucleic acid extraction process to be simpler by removing inhibitory substances using an absorbent (resin type) in an absorption chamber (200) rather than the conventional concept of capturing and concentrating nucleic acids using a column method or magnetic beads and then extracting nucleic acids again from the beads, thereby dramatically speeding up and simplifying the nucleic acid extraction process.
  • the nucleic acids can be automatically separated from a buffer (washing solution) in a waste collection and nucleic acid separation chamber (500) by using the difference in pressure.
  • the cartridge (2000) is mounted on the nucleic acid extraction device (3000) to perform the nucleic acid extraction process. Air is supplied into the interior of the injection chamber (100) through the docking portion (11) of the cartridge (2000) from a pump connected to the nucleic acid extraction device (3000).
  • 2 is connected to 3.
  • the air flowing to 2 moves to 3 on the opposite side.
  • the air moves from 3 to 4, moves to the opposite side again through 5, which is connected to 4, and rises to 6, and then is supplied to the injection chamber (100).
  • the sample received in the injection chamber (100) is discharged to 7 by air pressure.
  • the sample moves to 8 comes out to 9 on the opposite side, and moves from 9 to 10 along the tube-shaped path.
  • the sample introduced to 10 comes out to 11 on the opposite side, flows into the washing liquid storage chamber (400), and then flows. At this time, the washing liquid stored in the washing liquid storage chamber (400) moves to No. 12 due to the movement pressure of the sample and is discharged to No.
  • Nos. 13 and 14 are connected by a tube-shaped path.
  • the washing liquid passing through No. 14 flows again into No. 15 on the opposite side and washes the resin contained in the first, second, and third absorption chambers (210, 220, 230) while passing through the absorption chamber (200).
  • the washing liquid passing through the absorption chamber (200) is discharged to No. 16 and passes through Nos. 17 and 18.
  • the washing liquid passes through Nos. 19 and 20 and flows into No. 21.
  • Nos. 17 and 18 are connected by a tube-shaped path.
  • the washing liquid introduced into No. 21 flows downward and enters the waste collection and nucleic acid separation chamber (500).
  • the washing liquid first flows downward through the washing liquid collection channel (520) to fill the waste chamber (510), and when the waste chamber (510) is filled with the washing liquid, the nucleic acid (nucleic acid to which the inhibitor is adsorbed in the absorption chamber (200) and which is discharged from the absorption chamber (200)) flowing following the washing liquid flows toward No. 23 as pressure is applied by the narrow microchannel (550) of No. 22.
  • the nucleic acid discharged through No. 23 exits through No. 24 on the opposite side and flows into the nucleic acid chamber (300) through No. 25 to be extracted.
  • the user can capture the nucleic acid extracted through No. 26 using a pipette (330), etc.
  • PCR cycle is defined as the following three steps as one cycle.
  • the above three steps mean: 1) a denaturing step of heating a sample solution containing double-stranded DNA to a specific temperature, for example, about 95°C, to separate the double-stranded DNA into single-stranded DNA; 2) an annealing step of providing an oligonucleotide primer having a sequence complementary to a specific base sequence to be amplified in the sample solution after the denaturing step, and cooling the solution together with the separated single-stranded DNA to a specific temperature, for example, 55°C, to bind the primer to the specific base sequence of the single-stranded DNA to form a partial DNA-primer complex; and 3) an extension (or amplification) step of forming double-stranded DNA based on the primer of the partial DNA-primer complex by maintaining the sample solution at an active temperature of DNA polymerase, for example, 72°C, after the annealing step, by DNA polymerase.
  • a specific temperature for example, about 95°C
  • PCR exponentially amplifies target nucleic acids having a specific base sequence by repeating the above three steps several times to the extent that the detection of a specific base sequence is possible.
  • CT value of 29.56 means that the average number of repetitions is 29.56.
  • the sample prepared using the conventional dissolution buffer contained an inhibitor, so even if the above three steps for PCR were performed, the amplification of a specific base sequence was not achieved due to the inhibitor.
  • Table 2 when the sample was passed through an anion exchange resin, a chelating resin, and a cation exchange resin and the three steps for PCR were repeated, it was confirmed that a specific base sequence was amplified to a detectable level.
  • the absorption chamber was configured as a three-stage chamber with resin layers including an anion exchange resin, a chelating resin, and a cation exchange resin from the upper side to the lower side, it was confirmed that the number of PCR cycle repetitions for detecting a specific base sequence was the shortest. That is, when the absorption chamber is configured to sequentially pass through the three-stage resin layers, the amplification of a specific base sequence can be completed quickly.
  • the absorption chamber (200) is sequentially arranged with first, second, and third absorption chambers (210, 220, 230) including a cathode-bearing anion exchange resin, a chelate resin, and a cathode-bearing cation exchange resin.
  • the absorption chamber (200) may be formed by selecting at least one of the first, second, and third absorption chambers (210, 220, 230). That is, the absorption chamber may be formed by a combination of at least one, two, or three layers.
  • the volume ratio of the anion exchange resin, the chelate resin, and the cation exchange resin formed of spherical resin particles included in the first, second, and third absorption chambers (210, 220, and 230) is substantially 1:1:1.
  • the flow rate of the sample penetrating the inside of the absorption chamber is adjusted to 15 ⁇ l/sec or less.
  • the flow rate of the sample penetrating the inside of the absorption chamber is adjusted to 15 ⁇ l/sec or less
  • the flow rate of nucleic acid discharged to the lower part of the absorption chamber is adjusted to 15 ⁇ l per second or less, and the inhibitor can be sufficiently absorbed into the absorbent of the spherical particles while the sample passes through the first, second, and third absorption chambers (210, 220, and 230).
  • the flow rate of nucleic acid discharge exceeds 15 ⁇ l per second, the movement speed of the sample becomes fast, so that the inhibitor cannot be sufficiently absorbed.
  • the amount of nucleic acid collected in the nucleic acid chamber (300) can be secured substantially the same as the amount of nucleic acid injected into the injection chamber (100).
  • the inhibitor contained in the sample is removed and the flow rate substantially discharged to the lower part of the absorption chamber (200) is maintained at approximately 500 ⁇ l, and the inside is obtained in a state where the inhibitor is removed and only the nucleic acid is contained. This significantly improves the secured flow rate of the sample containing only the nucleic acid compared to the prior art.
  • the double-sided chip for nucleic acid extraction for PCR testing, the cartridge including the same, and the nucleic acid extraction device according to the embodiment of the present invention effectively remove foreign substances from the biological sample while moving the biological sample within a single chip without using additional equipment, and easily separate the nucleic acid and buffer, thereby providing the effect of quickly and easily extracting the nucleic acid.
  • the present invention excludes the use of additional equipment such as a centrifuge for nucleic acid separation, so that it is easy to carry and move to the site, and can improve the convenience of the user.

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  • Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne une puce double face pour l'extraction d'acide nucléique pour un test de PCR, une cartouche la comprenant, et un dispositif d'extraction d'acide nucléique l'utilisant. La cartouche comprenant la puce pour l'extraction d'acide nucléique comprend : une partie corps en forme de plaque comprenant une chambre d'absorption pour absorber et éliminer, à partir d'un échantillon, des inhibiteurs qui inhibent les performances de PCR ; un couvercle avant accouplé à un côté de la partie corps ; et un couvercle arrière accouplé au couvercle avant avec la partie corps intercalée entre ceux-ci, l'échantillon passant à travers la chambre d'absorption de sorte qu'un acide nucléique soit séparé de l'échantillon.
PCT/KR2024/010470 2023-07-21 2024-07-19 Puce double face pour extraction d'acide nucléique pour test de pcr, cartouche la comprenant, et dispositif d'extraction d'acide nucléique l'utilisant Pending WO2025023652A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20230095489 2023-07-21
KR10-2023-0095489 2023-07-21
KR10-2024-0095536 2024-07-19
KR1020240095536A KR20250015981A (ko) 2023-07-21 2024-07-19 Pcr 검사를 위한 핵산 추출용 양면칩, 이를 포함하는 카트리지, 및 이를 이용한 핵산추출장치

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WO2025023652A1 true WO2025023652A1 (fr) 2025-01-30

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020019060A1 (en) * 1999-05-28 2002-02-14 Cepheid Device for analyzing a fluid sample
KR20080022035A (ko) * 2006-09-05 2008-03-10 삼성전자주식회사 핵산 검출을 위한 원심력 기반의 미세유동장치 및 이를포함하는 미세유동시스템
KR102027101B1 (ko) * 2018-01-05 2019-10-02 성균관대학교산학협력단 시료의 농축 및 정제를 위한 미세유체 칩 및 전처리 방법
KR20200120068A (ko) * 2019-04-11 2020-10-21 (주)바이오니아 중합효소 연쇄반응 시스템
KR102177634B1 (ko) * 2018-02-05 2020-11-11 주식회사 진시스템 분자진단용 전처리 키트

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020019060A1 (en) * 1999-05-28 2002-02-14 Cepheid Device for analyzing a fluid sample
KR20080022035A (ko) * 2006-09-05 2008-03-10 삼성전자주식회사 핵산 검출을 위한 원심력 기반의 미세유동장치 및 이를포함하는 미세유동시스템
KR102027101B1 (ko) * 2018-01-05 2019-10-02 성균관대학교산학협력단 시료의 농축 및 정제를 위한 미세유체 칩 및 전처리 방법
KR102177634B1 (ko) * 2018-02-05 2020-11-11 주식회사 진시스템 분자진단용 전처리 키트
KR20200120068A (ko) * 2019-04-11 2020-10-21 (주)바이오니아 중합효소 연쇄반응 시스템

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