WO2023075045A1 - Puce sur support de papier pour réguler l'écoulement de fluide au moyen de barrières de cire - Google Patents

Puce sur support de papier pour réguler l'écoulement de fluide au moyen de barrières de cire Download PDF

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WO2023075045A1
WO2023075045A1 PCT/KR2022/004691 KR2022004691W WO2023075045A1 WO 2023075045 A1 WO2023075045 A1 WO 2023075045A1 KR 2022004691 W KR2022004691 W KR 2022004691W WO 2023075045 A1 WO2023075045 A1 WO 2023075045A1
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pad
sample
reaction
buffer
detection
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PCT/KR2022/004691
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English (en)
Korean (ko)
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김종철
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주식회사 에이아이더뉴트리진
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Publication of WO2023075045A1 publication Critical patent/WO2023075045A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/069—Absorbents; Gels to retain a fluid
    • 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
    • B01L2300/12—Specific details about materials
    • B01L2300/126—Paper
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/101—Temperature

Definitions

  • the present invention relates to a paper chip that controls fluid flow using a wax barrier, and more particularly, to a paper chip that includes a plurality of wax barriers and can control fluid flow in a pad.
  • the current universal molecular diagnosis method uses real-time PCR, which is the most common because of its speed, but it is difficult to use easily because it requires large and expensive equipment for on-site diagnosis or primary and secondary medical institutions.
  • three steps are largely required: sample preparation, nucleic acid amplification reaction, and detection.
  • nucleic acid amplification reaction and detection can be reproduced simultaneously by real-time PCR equipment, The preprocessing problem still remains.
  • lab-on-paper technology refers to a technology based on an integrated system that performs sample pretreatment, isothermal amplification, detection, and analysis steps on a single chip. Since all reactions can be automated and quickly performed with small paper and a chip structure embedded in the paper, it has the advantage of being independent of the location such as the detection site.
  • the paper chip manufactured by the conventional wrap-on-paper technology includes a single wax barrier, and when both sides of the pad are wet, the single wax barrier cannot block the flow of fluid.
  • Patent Document 1 KR 10-1662802 B1
  • a sample pad accommodating a biological sample
  • a buffer pad disposed separately from the sample pad and accommodating a rehydration buffer
  • a first connection pad disposed above the sample pad and connecting the sample pad and the reaction pad;
  • an initiator pad disposed above the buffer pad and connecting the buffer pad and the reaction pad;
  • reaction pad disposed below the first connection pad and the initiator pad, including a primer capable of specifically binding to a target nucleic acid and a reagent for an isothermal amplification reaction (LAMP), wherein an isothermal amplification reaction occurs;
  • LAMP isothermal amplification reaction
  • a blocking pad disposed above the reaction pad to maintain the reaction temperature and block evaporation of the sample
  • a detection pad disposed under the second connection pad and obtaining target nucleic acid amplified from an isothermal amplification reaction coupled with the gold nanoparticles;
  • An absorption pad disposed on a side of the detection pad and absorbing the remaining sample
  • a heating pad disposed below the sample pad, the initiator pad, the reaction pad, and the second connection pad,
  • the initiator pad and the second connection pad provide a structure for detecting multiple nucleic acids including a plurality of wax barriers.
  • sample pad and the first connection pad and the buffer pad and the initiator pad; reaction pad; a second connection pad; detection pad; and the absorbent pads are sequentially disposed at least partially in contact with each other, and a structure for detecting multiple nucleic acids is provided.
  • Another aspect provides a kit for diagnosing a disease or fungal infection, including the structure for detecting multiple nucleic acids.
  • Another aspect provides a method for providing information for diagnosing a disease or fungal infection using the structure for detecting multiple nucleic acids.
  • the disease or fungal infection diagnosis system of the present invention is based on lab-on-paper chip technology, and when the disease or fungal infection diagnosis system of the present invention is used, it moves to the reaction pad without separate nucleic acid purification.
  • a nucleic acid material can be purified and immediately applied to an amplification reaction, and a plurality of target nucleic acids can be simultaneously detected and related diseases can be diagnosed by applying one sample.
  • the structure for detecting nucleic acids of the present invention includes a sample pad 120, a first connection pad 131, a buffer pad 121, an initiator pad 132, a reaction pad 140, and a heating pad 141 ), a blocking pad 142, a second connection pad 150, a detection pad 160, an absorption pad 170, and a housing 110 as components.
  • the sample pad 120 accommodates a sample containing a nucleic acid material.
  • the sample is separated from the human body and includes blood, serum, plasma, saliva, sweat, urine, cell culture fluid, tissue suspension, etc., but is not limited thereto.
  • the sample may be mixed with a cell lysis buffer, and, if necessary, after mixing with the cell lysis buffer, it may be further purified by a commonly known method such as centrifugation, filtration, or precipitation.
  • the sample does not include a step of purifying the sample to be applied to the sample pad other than the structure for detecting nucleic acid after being mixed with the cell lysis buffer.
  • the cell lysis buffer may contain Tris (tris (hydroxymethyl) aminomethane) at 5 mM to 80 mM, 5 mM to 50 mM, or 10 mM to 50 mM. . Tris may specifically be Tris-HCl, and can reduce rapid pH fluctuations as a buffering agent in a cell lysis buffer.
  • the cell lysis buffer may have a pH of 8.0 to 9.0. When the pH is less than 8.0, the stability of the nucleic acid material may be reduced or the rate of migration may be reduced.
  • the cell lysis buffer may contain potassium chloride (KCl) at 5 mM to 50 mM, 5 mM to 40 mM, or 10 mM to 20 mM.
  • KCL potassium chloride
  • a high concentration of potassium chloride exceeding 50 mM is helpful for cell lysis, but reduces the aqueous solubility of the eluted nucleic acid material, which may require the addition of a large amount of additional buffer or increase the time taken to move to the reaction pad.
  • potassium chloride is less than 5 mM, cells may not be properly lysed.
  • the cell lysis buffer may contain magnesium sulfate (MgSO 4 ) at 1 mM to 30 mM, 1 mM to 20 mM, or 2 mM to 16 mM. It has been confirmed that the stability and migration speed of nucleic acid substances are increased when an appropriate amount of magnesium sulfate is included, and since it does not affect the viscosity, it does not disturb the flow of the fluid, which is advantageous in pre-processing the sample for paper chip analysis.
  • MgSO 4 magnesium sulfate
  • the cell lysis buffer may contain ammonium sulfate ((NH 4 ) 2 SO 4 ) at 5 mM to 50 mM, 5 mM to 40 mM, or 10 mM to 20 mM. High concentrations of ammonium sulfate, greater than 50 mM, may precipitate cell lysates, and pH may become unstable when ammonium sulfate is less than 5 mM.
  • ammonium sulfate (NH 4 ) 2 SO 4 ) at 5 mM to 50 mM, 5 mM to 40 mM, or 10 mM to 20 mM.
  • High concentrations of ammonium sulfate, greater than 50 mM may precipitate cell lysates, and pH may become unstable when ammonium sulfate is less than 5 mM.
  • the cell lysis buffer may contain 0.01 mg/ml to 0.1 mg/ml or 0.03 mg/ml to 0.07 mg/ml of proteolytic enzyme.
  • the proteolytic enzyme degrades the polymer protein so that the polymer protein does not block the pores of the substrate or paper, which is the path for nucleic acid to move, and increases the stability of the nucleic acid material by inhibiting the activities of RNase and DNase.
  • the proteolytic enzyme may be proteinase K.
  • the surfactant may be TritonX-100 or Tween20 (polysorbate 20), 0.01 w/w% to 0.2 w/w%, preferably 0.05 w/w% to 0.1 w based on the weight of the cell lysis buffer. Can be included as /w%.
  • the cell lysis buffer may be used in a ratio of 1:1 to the volume of the sample.
  • the cell lysis buffer may not contain glycerol. Glycerol is sometimes added to prevent protein precipitation, but glycerol increases the viscosity, reduces the fluidity of the cell lysate, and may interfere with the movement of nucleic acid materials.
  • the cell lysis buffer may not contain a reducing agent.
  • Reducing agents such as dithiothreitol (DTT) and mercaptoethanol, help to denature proteins and increase the solubility of cell lysates, but interfere with fluorescence or detection reactions when present in the solution flowing into the paper chip. It can be.
  • the heating pad is heated at a temperature of 60 to 80 ° C for 1 to 5 minutes, preferably for 5 minutes, so that the sample pad contains viruses and epithelial cells. Promote dissolution of the sample.
  • the cell lysis composition promotes cell lysis in the sample pad 120 made of a polysulfone membrane (eg, Vivid GF) or a nitrocellulose membrane, and makes it easier to detect nucleic acids.
  • the polysulfone membrane and the nitrocellulose membrane may form a structure in which two or more are laminated.
  • the polysulfone membrane may be asymmetric, and the polysulfone membrane and the nitrocellulose membrane may be porous materials having pores of 0.5 ⁇ m to 1 ⁇ m. It is advantageous that the pores are rather large.
  • Many biological samples have viscosity, and in particular, when the sample is treated with a composition for cell dissolution, the viscosity may increase significantly as nucleic acid materials and proteins are eluted out of the cells. Therefore, it is preferable that the pore size of the sample pad has an appropriate size to rapidly absorb the sample.
  • lateral flow method refers to a method in which a sample is flowed from an application point to a target point by a capillary phenomenon or a diffusion phenomenon in a horizontal direction without using gravity. Since the cell lysate contains a large amount of hydrolytic enzymes capable of degrading nucleic acid material, the yield of nucleic acid material may be reduced if the cell lysate remains in the migration path for a long time. Therefore, in order to be applied to a structure for detecting nucleic acid in a lateral flow type, the flow rate should be excellent and the cell lysate should not precipitate and block the movement path while the sample is moving.
  • composition of the cell lysis buffer of the present invention does not precipitate cell lysates or nucleic acid materials even if it does not contain glycerol or a reducing agent, and can transfer nucleic acid materials laterally to the reaction pad in high yield.
  • the first connection pad 131 partially contacts the sample pad, is disposed above the sample pad, and connects the sample pad and the reaction pad with a structure having a relatively narrow width compared to the sample pad.
  • the first connection pad may be made of a cellulose membrane and have pores of 0.005 ⁇ m to 0.015 ⁇ m.
  • the buffer pad 121 is a pad to which the rehydration buffer is applied dropwise, and serves to apply water pressure to the structure.
  • the buffer pad may be disposed separately from the sample pad in order to minimize the movement of cell lysis debris such as proteins to the reaction pad and induce the movement of only the isothermal amplification reactants to the detection pad by applying water pressure after the reaction is completed.
  • the rehydration buffer applied to the buffer pad is an additional buffer, for example, 5 mM to 80 mM Tris-HCl, 20 mM to 70 mM potassium chloride, 0.5 mM to 5 mM magnesium sulfate, 1 mM to 30 mM ammonium sulfate, and 0.01 mM It may be an isothermal buffer containing w/w% to 0.2 w/w% Tween® 20 to TritonX-100 and having an acidity of pH 8.0 to 9.0 or a phosphate buffer (50 mM Na 2 HPO 4 , pH 7.2).
  • the isothermal buffer may more specifically include 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , and 0.1% Tween® 20, and may have a pH of 8.8.
  • the addition buffer may not contain a protease, glycerol, or a reducing agent.
  • the buffer pad is a porous material having pores of 0.5 ⁇ m to 1 ⁇ m to sufficiently receive the rehydration buffer, and is made of cotton, flax, paper, nitrocellulose, cellulose acetate, glass fiber, polysulfone, polyacrylic, polynitrile, polypiperazine, polyamide, polyethersulfone, polyvinylidene fluoride, polyethyleneimine, polydimethylsiloxane or mixtures thereof.
  • the initiator pad 132 is disposed above the buffer pad and has a relatively narrow width compared to the buffer pad, and connects the buffer pad and the reaction pad.
  • the initiator pad may be made of a cellulose membrane and have pores of 0.005 ⁇ m to 0.015 ⁇ m.
  • An initiator pad at a portion in contact with the reaction pad may be coated with low melting point agarose or wax.
  • the initiator pad may include a wax barrier, and the wax barrier may include a first wax barrier and a second wax barrier.
  • the wax barrier is formed to a certain thickness on one side of the pad. More specifically, a first wax barrier may be formed to have a certain thickness in a direction perpendicular to a direction in which the rehydration buffer solution of the initiator pad is absorbed, and a second wax barrier may be formed at opposite positions spaced apart from each other at regular intervals. .
  • the present invention is characterized in that a plurality of wax barriers are formed on the initiator pad, and the wax barriers are spaced apart and disposed at regular intervals.
  • the first wax barrier has a width of 1.5 to 1.75 mm
  • the second wax barrier has a width of 0.1 to 0.3 mm.
  • the initiator pad of the present invention blocks the rehydration buffer solution of the buffer pad by the first wax barrier and the second wax barrier, and then heats the initiator pad at the end of the reaction in the reaction pad to rehydrate.
  • the buffer solution By flowing the buffer solution sideways, the nucleic acid amplified in the reaction pad can flow to the detection pad.
  • a heating pad 141 for heating may be disposed under the initiator pad. When heated at the end of the isothermal amplification, the isothermally amplified product from the reaction pad is easily moved to the detection pad. The heating may be performed at 60 to 80° C. for 1 minute to 5 minutes, preferably for 2 minutes.
  • the reaction pad is a component corresponding to the paper chip in lab-on-paper, and isothermal amplification reagents including dNTP, DNA polymerase, reverse transcriptase, fluorescent marker, isothermal amplification reaction buffer, etc. for amplification reaction are fixed thereto. Therefore, the solution containing the nucleic acid material penetrates into the reaction pad by the additional buffer applied to the sample pad and is wetted, and the contact material between the isothermal amplification reagent and the sample moves to the reaction pad and is heated by the heating pad at the bottom of the reaction pad. When heated to 60 to 70 ° C., an isothermal amplification reaction or a reverse transcription isothermal amplification reaction occurs.
  • the isothermal amplification reagent is specifically dNTP (1.4mM, dATP, dCTP, dGTP and dTTP), isothermal amplification buffer (1X, 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 2mM MgSO 4 , and 0.1% Tween-20, pH7.5), and Bst 3.0 DNA polymerase (320 U/ml), which are mixed in the reaction pad and then dried or applied to the surface of the reaction pad in powder form, for example, It may be fixed by heating in an oven at about 35 to 40 ° C. for about 30 minutes.
  • the reaction pad 140 may partially come into contact with the first connection pad and the initiator pad, and may be disposed under and sideways of the first connection pad and the initiator pad.
  • a heating pad 141 may be disposed below the reaction pad 140 to heat to a temperature at which an isothermal amplification reaction may occur.
  • the heating pad may include a hot wire or a hot plate for heating. The heating may be performed at 60 to 70°C, preferably 60 to 65°C for 20 minutes to 1 hour, preferably 20 minutes to 30 minutes.
  • the reaction pad may have a blocking pad 142 disposed above the reaction pad to serve as a blocking function.
  • the isothermal amplification reaction temperature is maintained by a blocking pad laminated to a series of arranged pads, and evaporation of reagents is blocked to increase reaction efficiency.
  • the blocking pad may be a non-porous membrane or a structure capable of blocking the reaction pad from outside air.
  • ⁇ M) and outer primers (F3 and B3, 0.2 ⁇ M) can be immobilized.
  • the concentration of the primers is based on the volume of the well, and the concentration of the primer set in the well can be changed while maintaining the concentration ratio between the respective primers.
  • An isothermal amplification reaction can occur more intensively by the presence of wells in the reaction pad.
  • the well may have a hydrogel layer formed at the bottom and the primer set fixed to the hydrogel layer.
  • primer sets that specifically bind to different target nucleic acids may be immobilized in each well.
  • the hydrogel layer containing the primer may be formed, for example, by the following method.
  • UV-light crosslinkable poly(ethylene glycol) diacrylate PEGDA, Sigma-Aldrich, MW700
  • PEG poly(ethylene glycol)
  • PEG poly(ethylene glycol)
  • MW600 poly(ethylene glycol)
  • PBS buffer pH7.5
  • the poly(ethylene glycol) is preferably included to increase the porosity of the hydrogel microparticles.
  • the hydrogel solution is applied to the inner surface of each well of the reaction pad and exposed to UV (360 nm wavelength, 35 mJ/cm 2 ) for 1 minute to form a hydrogel coating layer. Since the hydrogel layer has pores, an amplification reaction may occur intensively in the pores by binding to primers in the hydrogel layer.
  • any one of the forward and reverse primers in the primer set is Cy3, Cy5, TAMRA, TEX, TYE, HEX, FAM, TET, JOE, MAX, ROX, VIC, Cy3.5, Texas Red, Cy5.5, TYE, BHQ , Iowa Black RQ, and may be labeled with one or more fluorescent markers selected from the group consisting of IRDye.
  • the fluorescent marker may be labeled differently for each target nucleic acid in order to independently detect the target nucleic acid.
  • the other one of the forward and reverse primers may be biotin-linked. Since biotin can bind to streptavidin, it exists in a form bound to the amplified target nucleic acid and passes through the second connection pad, binds to streptavidin on the surface of the gold particle and is detected when captured by the detection pad Make the results visible. Biotin may be designed to be present at opposite positions of a detector and a detector in the amplified target nucleic acid. For example, when a detector is bound to the 5' end of a forward primer, biotin may be bound to the 5' end of a reverse primer. there is.
  • the reaction pad is made of a cellulose acetate membrane material, and has a pore size of 0.001 ⁇ m to 0.005 ⁇ m, preferably 0.005 ⁇ m, so that the nucleic acid can remain and sufficiently isothermally amplified while allowing the sample and the additional buffer to flow freely.
  • the reaction pad may include 40 mM to 50 mM sucrose, 0.001 to 0.01% Triton X-100, and 0.1 w/w% to 0.3 w/w% glycerol. This can increase their storage stability when the isothermal amplification reagent and primer set are exposed to moisture or oxygen.
  • the storage stability may mean that it can be stored for 3 weeks or more without degradation products or by-products at 25 ° C to 30 ° C.
  • the second connection pad 150 may partially come into contact with the reaction pad and may be disposed above and sideways of the reaction pad.
  • the second connection pad 150 includes gold nanoparticles, and the gold nanoparticles combine with the nucleic acid amplified in the reaction pad and move to the detection pad.
  • the gold nanoparticles may preferably have streptavidin immobilized on the surface.
  • the second connection pad is cotton, wool, paper, nitrocellulose, glass fiber, polysulfone, polyacrylic, polynitrile, polypiperazine, polyamide, polyethersulfone, polyethersulfone, As a porous material of vinylidene fluoride, polyethyleneimine, polydimethylsiloxane, or a mixture thereof, it may have a pore size of 0.01 ⁇ m to 0.05 ⁇ m, preferably, 0.05 ⁇ m.
  • the second connection pad may have a cellulose material, and the second connection pad at a portion in contact with the reaction pad may be coated with low melting point agarose or wax.
  • a wax barrier may be formed on the second connection pad in the same manner as the previous initiator pad. More specifically, the wax barrier may be formed of a first wax barrier and a second wax barrier. The wax barrier is formed to a certain thickness on one side of the pad. More specifically, a first wax barrier may be formed to have a certain thickness in a direction perpendicular to a direction in which the isothermal amplification reactant of the second connection pad is absorbed, and a second wax barrier may be formed at opposite positions spaced apart from each other at regular intervals. .
  • the second connection pad blocks the flow of the isothermal amplification reactant through a single wax barrier, but if both sides of the second connection pad become wet, a single wax barrier may not block the fluid flow. This is a phenomenon caused by the merging of the meniscus of the fluid by capillary action.
  • the present invention is characterized in that a plurality of wax barriers are formed on the second connection pad, and the wax barriers are spaced apart and disposed at regular intervals.
  • the first wax barrier has a width of 1.5 to 1.75 mm
  • the second wax barrier has a width of 0.1 to 0.3 mm.
  • the flow of the isothermal amplification reactant may be blocked by the first wax barrier and the second wax barrier.
  • the movement of the isothermal amplification reactant in the reaction pad is blocked by the first wax barrier and the second wax barrier, and then the coating melts at the time of heating the second connection pad, so that the isothermal amplification reactant moves to the side. It is possible to prevent the loss of unreacted genetic material in the sample by moving back to .
  • a heating pad 141 for heating may be disposed under the second connection pad.
  • the heating may be performed at 60 to 80° C. for 1 minute to 5 minutes, preferably for 2 minutes.
  • the detection pad 160 may partially come into contact with the second connection pad and may be disposed below and on the side of the second connection pad.
  • a receptor that can bind to the detector is fixed to the detection pad 160 .
  • the receptor may be an antibody, protein, or fragment thereof capable of specifically binding to a detector.
  • the detection pad includes a plurality of detection regions, and the detection regions may be divided into lines or wells.
  • each receptor is independently immobilized.
  • a solution containing a receptor and EDC (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide) or NHS (N-hydroxysulfosuccinimide) may be applied.
  • EDC 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide
  • NHS N-hydroxysulfosuccinimide
  • the detection pad is nitrocellulose and may have a pore size of 0.001 ⁇ m to 0.005 ⁇ m, preferably 0.005 ⁇ m, so that the sample can move laterally to the absorption pad.
  • a heating pad 141 may be disposed below the sample pad, the initiator pad, the reaction pad, and the second connection pad.
  • the heating pad is a metal plate having thermal conductivity, and may be made of a material such as iron, stainless steel, aluminum, silver, or copper.
  • the heating pad may be connected with a heating wire, and each heating wire may be independently connected to the sample pad, the reaction pad, and the heating pad under the second connection pad to control the heating pad.
  • the sample pad, the first connection pad, the initiator pad, the reaction pad, the second connection pad, the detection pad, and the absorption pad may have housings 110 at upper and lower ends so that the sample or buffer is not lost, and the entire structure is a housing. It may be surrounded by a case of a support or non-porous material that can be defined as.
  • the absorbent pad 170 absorbs a sample and a buffer to block reverse flow and contribute to induce lateral fluidity.
  • the absorbent pad is a porous material, such as cotton, flax, paper, nitrocellulose, cellulose acetate, glass fiber, polysulfone, polyacrylic, polynitrile, polypiperazine, polyamide, polyethersulfone, polyvinylidene fluoride, and polyethyleneimine. , polydimethylsiloxane or mixtures thereof.
  • the absorbent pad is preferably a glass fiber and may have a pore size of 0.1 to 0.5 ⁇ m.
  • the sample pad, the first connection pad, the buffer pad, and the initiator pad; reaction pad; a second connection pad; detection pad; and the absorbent pads are sequentially disposed laterally in partial contact with each other. Specifically, the sample pad, the first connection pad, the buffer pad, and the initiator pad are separated based on the reaction pad and disposed on one side of the reaction pad, followed by the second connection pad, detection pad, and The absorbent pads are sequentially disposed.
  • nucleic acid materials can be purified by filtering cell lysate in addition to nucleic acid materials while the sample reaches the absorption pad due to the arrangement and characteristics of each component of the structure, and the moving direction of the sample is different from the ground. Lateral fluidity, which is a parallel direction, can be realized.
  • nucleic acids exist in the form of proteins such as histones, polymerases, nucleases, and transcription factors.
  • proteins lose their binding to nucleic acids by surfactants such as the cell lysis composition, but by the time the cell lysate reaches the reaction pad by the addition of distilled water or added buffer so that the cell lysate can move to the reaction pad, the surfactant is diluted, allowing the external environment to regain the protein's charge.
  • the protein may bind to the nucleic acid material again to reduce the contact area with the polymerase or interfere with the amplification reaction. Therefore, it is preferable that most cellular components capable of binding to nucleic acids are purified and removed from the reaction pad.
  • a sample for which nucleic acid is to be detected may include mixing with a cell lysis buffer prior to application to the sample pad.
  • the nucleic acid material present in the cells can be eluted, and thus the amount of the nucleic acid material detectable in the sample can be increased.
  • Tris-HCl Tris-HCl, potassium chloride 5 mM to 50 mM, magnesium sulfate 1 mM to 30 mM, ammonium sulfate 5 mM to 50 mM, protease 0.01 mg/ml to 0.1 mg/ml, TritonX- 100 or Tween20 0.01 w/w% to 0.2 w/w%, by adding a cell lysate mixed with a cell lysis buffer having a pH of 8.0 to 9.0 dropwise and operating a heating pad below the sample pad to heat the sample pad Increase the efficiency of sample dissolution. By heating the heating pad at a temperature of 60 to 80° C. for 2 minutes, dissolution of the sample including viruses and epithelial cells in the sample pad is promoted.
  • a buffer solution may be applied to the buffer pad.
  • the buffer serves to purify the nucleic acid material while allowing the nucleic acid material to move to the reaction pad.
  • the buffer serves to purify the nucleic acid material while allowing the nucleic acid material to move to the detection pad. Since the buffer solution contains an appropriate amount of a buffering component, it is possible to prevent precipitation of proteins or nucleic acids due to rapid changes in salinity or pH that may occur when distilled water is added.
  • Each pad of the structure for detecting nucleic acids of the present invention is made of a porous material having small pores so that nucleic acids can be purified while moving laterally. Therefore, if the protein or nucleic acid material is complexed or aggregated to block the pores, the movement speed of the sample may decrease and the yield of the nucleic acid material may decrease.
  • the addition buffer is, for example, 5 mM to 80 mM Tris-HCl, 20 mM to 70 mM potassium chloride, 0.5 mM to 5 mM magnesium sulfate, 1 mM to 30 mM ammonium sulfate, and 0.01 w/w% to 0.2 w/w % Tween® 20 to TritonX-100 and an acidity pH of 8.0 to 9.0 or a phosphate buffer (50 mM Na 2 HPO 4 , pH 7.2).
  • the isothermal buffer may more specifically include 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , and 0.1% Tween® 20, and may have a pH of 8.8.
  • the addition buffer may not contain a protease, glycerol, or a reducing agent.
  • the heating pad under the reaction pad may be heated to 60 to 65° C., and allowed to stand for 20 minutes to 1 hour, preferably 20 to 30 minutes, while maintaining the temperature.
  • Diseases that can be diagnosed using the multi-nucleic acid detection structure are those in which a specific gene can be used as a cause or indicator of a disease, for example, metabolic diseases such as obesity, hypertension, diabetes, genetic diseases, cancer, etc.
  • metabolic diseases such as obesity, hypertension, diabetes, genetic diseases, cancer, etc.
  • viruses or bacteria are related to infectious diseases, and bacteria that can cause fungal infections include bacteria, protozoa, parasites, fungi, etc., but are not limited thereto.
  • the present invention is characterized by controlling the flow of fluid using a pad including a plurality of wax barriers, enabling highly accurate detection by application of a sample.
  • the sample is applied once and the detection is performed at once without separately performing sample preparation, nucleic acid amplification reaction, and detection. can be done Since each step is not performed separately, the entire process is simplified, and various samples or devices are not required, and it can be easily performed even without a related technician.
  • 1 is an example of the overall structure of the construct for detecting multiple nucleic acids of the present invention.
  • 3 is an experimental result of fluid flow control by a wax barrier according to an embodiment of the present invention.
  • FIG. 4 is a diagram illustrating the principle of obtaining target nucleic acids from the detection pad 160.
  • 5 is a side structural view of a part of the structure for detecting multiple nucleic acids of the present invention.
  • FIG. 6 is a perspective view of a part of the structure for detecting multiple nucleic acids of the present invention.
  • FIG. 7 is an enlarged structural diagram of the structure of the detection pad 160.
  • FIG. 8 is an example showing the appearance of the structure for detecting multiple nucleic acids of the present invention surrounded by a case.
  • FIG. 7 is an example showing the result of detecting SARS-CoV-2 from a blood sample.
  • the present invention relates to a sample pad accommodating a biological sample; a buffer pad disposed separately from the sample pad and accommodating a rehydration buffer; a first connection pad disposed above the sample pad and connecting the sample pad and the reaction pad; an initiator pad disposed above the buffer pad and connecting the buffer pad and the reaction pad; a reaction pad disposed under the first connection pad and the initiator pad, including a primer capable of specifically binding to a target nucleic acid and a reagent for an isothermal amplification reaction (LAMP), wherein an isothermal amplification reaction occurs; a blocking pad disposed above the reaction pad to maintain the reaction temperature and block evaporation of the sample; a second connection pad disposed above the reaction pad and to which gold nanoparticles are fixed; a detection pad disposed under the second connection pad and obtaining a target nucleic acid amplified from an isothermal amplification reaction coupled with the gold nanoparticles; and an absorption pad disposed on a side of the detection pad to absorb the remaining sample, and
  • the magnetic pad and the second connection pad include a first wax barrier and a second wax barrier, the first wax barrier has a width of 1.5 to 1.75 mm, and the second wax barrier has a width of 0.1 to 0.3 mm.
  • the first wax barrier and the second wax barrier block a stable fluid flow, and form a smooth fluid flow when the temperature is raised by the heating pad.
  • the sample pad 120 and buffer pad 121 0.01 ⁇ m cellulose was used as the first connection pad 131 or initiator pad 132
  • the reaction pad 140 was made of 0.005 ⁇ m cellulose acetate
  • the second connection pad 150 was made of 0.05 ⁇ m cellulose
  • the detection pad 160 was made of 0.005 ⁇ m nitrocellulose
  • the absorption pad 170 was made of 0.5 ⁇ m glass fiber.
  • the reaction pad 140 was prepared by making a pad by overlapping cellulose acetate films, immersing the pad in a solution containing 45 mM sucrose, 0.005 w/w% TritonX-100, and 0.2 w/w% glycerol, and drying. Then, a well was formed using a micro-drill, and a hydrogel layer including a primer set was formed on the bottom of the well.
  • hydrogel layer To form the hydrogel layer, first, based on the total volume of the hydrogel solution, UV-light crosslinkable poly (ethylene glycol) diacrylate (PEGDA, Sigma-Aldrich, MW700) 20% v / v, poly (ethylene glycol) ( Mix PEG, Sigma-Aldrich, MW600) 40% v/v and photoinitiator 2-hydroxy-2-methylpropiophenone (Sigma-Aldrich) 5% v/v and buffer (PBS buffer, pH7.5) 35% Then, a hydrogel solution was prepared by mixing each primer set therein. The poly(ethylene glycol) is preferably included to increase the porosity of the hydrogel microparticles. Then, the hydrogel solution was applied to the inner surface of each well of the reaction pad and exposed to UV (360 nm wavelength, 35 mJ/cm 2 ) for 1 minute to form a hydrogel coating layer.
  • PEGDA poly (ethylene glycol) diacrylate
  • PEG Poly (ethylene glycol)
  • dNTP 1.4mM, dATP, dCTP, dGTP and dTTP
  • isothermal amplification buffer (1X, 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 2mM MgSO 4 were added to the surface of the reaction pad 140.
  • 0.1% Tween-20, pH 7.5) and Bst 3.0 DNA polymerase (320 U/ml) were applied, and fixed by heating in an oven at about 38° C. for about 30 minutes.
  • the gold nanoparticles fixed on the second connection pad 150 were prepared as colloidal particles as follows. When the 0.1% HAuCl 4 solution starts to boil while stirring and heating, 0.5% sodium citrate solution is added to reduce the solution to form gold particles, and 1 mg of streptavidin per 100 ml of the gold particle solution is added to condense made it The condensate was precipitated by centrifugation at 10,000 g, dissolved in physiological saline (PBS) containing 0.1% BSA, and stored at an OD450 value of 10.
  • PBS physiological saline
  • the second connection pad 150 was manufactured as follows. Specifically, several layers of cellulose membranes were prepared and cut, 0.4 M Tris (pH 6.5), 0.2% Tween-20, 1% sodium caseinate, 0.1% sodium azide, and 0.05% Proclin. It was immersed in a solution prepared by 300 and kept wet. The prepared gold condensate was prepared by dialysis against a solution having the same composition as the above solution. Then, the cellulose membrane was treated with the dialyzed gold condensate and dried to complete the second connection pad 150.
  • the detection pad 160 is designated by stamping the detection area with polyethylene phthalate ink, and then stamped again with a solution containing an antibody capable of binding to a fluorescent marker such as FAM, HEX, and Cy5 thereon, and NHS (N- hydroxysulfosuccinimide) solution was applied and reacted to fix the antibody.
  • a fluorescent marker such as FAM, HEX, and Cy5
  • NHS N- hydroxysulfosuccinimide
  • a portion of the second connection pad 150 in contact with the reaction pad was coated with a 5% low melting point agarose (Lonza, NuSieve GTG Agarose) solution.
  • each structure is arranged as shown in FIG. 3, and the sample pad 120, the initiator pad 132, the reaction pad 140, and the second connection pad 150 are provided with heating pads at the lower ends.
  • a copper plate connected to a hot wire at 141 was disposed, and a polyacrylic film was disposed as a blocking pad 142 on top of the reaction pad.
  • Wax barriers of 1.5 mm/0.2 mm (first wax barrier/second wax barrier) and 1.75 mm/0.2 mm (first wax barrier/second wax barrier) were formed on 0.01 ⁇ m cellulose.
  • the method of forming the wax barrier was produced by printing to have the width as described above using a wax front, and drying in an oven at 130 degrees for 45 seconds so that the wax could sufficiently permeate.
  • a cellulose pad containing a dye solution was connected to the surface where the first wax barrier was located, and the fluid flow was checked.
  • Test Example 2 Virus detection using blood samples
  • Virus infection such as SARS-CoV-2 can be easily measured by extracting nucleic acid from a blood sample using the lab-on-paper nucleic acid detection structure of the present invention, amplifying the nucleic acid, and measuring fluorescence.
  • a set of primers capable of selectively binding to the N protein gene characteristic of SARS-CoV-2 or the Rdrp gene can be used.
  • one of the forward or reverse primers of each set is, for example, FAM, HEX, or Cy5 bound, and the other primer is biotin bound.
  • the amplified nucleic acid bound to the gold nanoparticle moves to the detection pad 160 by lateral flow, and the detector bound to the other side of the amplified nucleic acid is specific for FAM, HEX, or Cy5 immobilized on the detection pad 160.
  • the color of the detection area of the detection pad is changed to pink while binding to the receptor that can be bound to it.
  • the principle of binding of the target nucleic acid to the detection pad is illustrated in FIG. 4 .
  • a primer set that selectively binds to a gene characteristic of a virus with a high possibility of cross-detection with SARS-CoV-2 can be introduced and detected together as a negative control.
  • Test Example 3 Nucleic acid extraction and amplification using blood samples
  • Nucleic acids were extracted from blood samples using the construct for detecting nucleic acids in lab-on-paper of the present invention, and nucleic acids were amplified to confirm whether they could exhibit fluorescence.
  • human blood was purchased and prepared as whole blood from Innovative Research (IWB1K2E10ML, USA), and 18S rRNA primers as a positive control were purchased from Tocris (# 7325, USA). It was confirmed through the data sheet that the whole blood used was not infected with any virus or bacteria.
  • One test sample was prepared by mixing 1 ⁇ l of 0.1 pg/ ⁇ l of SARS-CoV-2 positive control from siTOOLs Biotech with 100 ⁇ l of the human blood.
  • F3 primer bound a detector to the 5' end
  • B3 primer bound biotin to the 5' end.
  • human 18s RNA (A) was introduced with FAM, and N gene (B) with Cy5 as a detector.
  • Lysis buffer (20 mM Tris HCl (pH 8.8), 15 mM MgSO 4 , 15 mM KCl, 15 mM (NH 4 ) 2 SO 4 , 0.1 50 ⁇ l of w/w% Tween20, 0.05 mg/ml proteinase K) was added and lightly tapped, followed by incubation at room temperature for about 5 minutes.
  • the heating pad 141 under the sample pad 120 operated at 60° C., it was slowly added dropwise to the sample pad 120 of the structure for detecting nucleic acid prepared in Preparation Example 1 within 5 minutes within 5 minutes, and the addition buffer ( 250 ⁇ l of 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , 0.1% Tween® 20, pH 8.8) was slowly added dropwise to the sample pad for about 2 minutes, followed by reaction. The heating pad under the pad was heated to 60 °C and reacted for 30 minutes.
  • the addition buffer 250 ⁇ l of 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , 0.1% Tween® 20, pH 8.8
  • the heating pad under the second connection pad 150 and the heating pad under the initiator pad 132 were heated to 80° C., and 250 ⁇ l of the addition buffer was slowly added dropwise to the buffer pad for 2 minutes and displayed on the detection pad. The color change of the detection zone was observed.
  • the present invention relates to a paper chip that controls fluid flow using a wax barrier, and more particularly, to a paper chip that includes a plurality of wax barriers and can control fluid flow in a pad.

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Abstract

La présente invention concerne une structure comprenant un tampon comportant une pluralité de barrières de cire, les barrières de cire pouvant réguler l'écoulement de fluide, et ainsi une efficacité de détection hautement précise pouvant être présentée. De plus, la présente invention concerne une structure apte à purifier et à détecter simultanément des acides nucléiques par application directe d'un échantillon, et est caractérisée par une structure à laquelle une technologie de laboratoire sur papier est appliquée de sorte que les étapes de prétraitement d'échantillon, d'amplification isotherme, de détection et d'analyse puissent être réalisées sur une seule puce, et l'échantillon est déplacé dans un mode à écoulement latéral de façon à être finalement relié à des mégadonnées génomiques associées à une maladie et à une infection souche, et ainsi si la maladie ou l'infection par souche est présente, peut être déterminé directement.
PCT/KR2022/004691 2021-10-29 2022-04-01 Puce sur support de papier pour réguler l'écoulement de fluide au moyen de barrières de cire WO2023075045A1 (fr)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
US20110124116A1 (en) * 1995-03-10 2011-05-26 Meso Scale Technology Llp Multi-array, multi-specific electrochemiluminescence testing
KR101662802B1 (ko) * 2015-09-22 2016-10-05 충남대학교산학협력단 유체의 이동속도 조절이 가능한 종이칩 및 그 제조방법
KR20170078057A (ko) * 2015-12-29 2017-07-07 광주과학기술원 다중 분자진단을 위한 칩 구조
JP2021100863A (ja) * 2019-12-24 2021-07-08 北越コーポレーション株式会社 チップ状電子部品用キャリアテープ台紙及びその製造方法
KR102310223B1 (ko) * 2021-07-21 2021-10-08 주식회사 에이아이더뉴트리진 랩온페이퍼칩을 포함하는 구조물에 적용하기 위한 세포용해용 조성물

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110124116A1 (en) * 1995-03-10 2011-05-26 Meso Scale Technology Llp Multi-array, multi-specific electrochemiluminescence testing
KR101662802B1 (ko) * 2015-09-22 2016-10-05 충남대학교산학협력단 유체의 이동속도 조절이 가능한 종이칩 및 그 제조방법
KR20170078057A (ko) * 2015-12-29 2017-07-07 광주과학기술원 다중 분자진단을 위한 칩 구조
JP2021100863A (ja) * 2019-12-24 2021-07-08 北越コーポレーション株式会社 チップ状電子部品用キャリアテープ台紙及びその製造方法
KR102310223B1 (ko) * 2021-07-21 2021-10-08 주식회사 에이아이더뉴트리진 랩온페이퍼칩을 포함하는 구조물에 적용하기 위한 세포용해용 조성물

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