WO2012047015A9 - 핵산 압타머 선별을 위한 멀티플렉스 미세 유체 장치 및 이를 이용한 핵산 압타머의 고속 선별방법 - Google Patents
핵산 압타머 선별을 위한 멀티플렉스 미세 유체 장치 및 이를 이용한 핵산 압타머의 고속 선별방법 Download PDFInfo
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- C12N15/1034—Isolating an individual clone by screening libraries
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- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- C12N2320/00—Applications; Uses
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- C12N2320/00—Applications; Uses
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- C12N2320/13—Applications; Uses in screening processes in a process of directed evolution, e.g. SELEX, acquiring a new function
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- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
- C12Q2525/10—Modifications characterised by
- C12Q2525/205—Aptamer
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- C12Q2541/00—Reactions characterised by directed evolution
- C12Q2541/10—Reactions characterised by directed evolution the purpose being the selection or design of target specific nucleic acid binding sequences
- C12Q2541/101—Selex
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Definitions
- the present invention relates to a multiplex microfluidic device for nucleic acid aptamer selection and a high-speed screening method of nucleic acid aptamer using the same. More specifically, the method for converting aptamer development for an existing single target into an improved multiplex platform A multiplex microfluidic device (SELEX lap-on-a-chip) and a high throughput selection method of nucleic acid aptamers characterized by the high throughput sequencing.
- Aptamers are single-stranded DNA or RNA molecules, and are small single-stranded oligonucleotides capable of specifically recognizing target materials with high affinity. Aptamers have been recognized as alternatives to antibodies because they can be used as a component of biosensors that can recognize molecules in detection and analysis systems. In particular, aptamers can be used as target molecules of various organic and inorganic substances, including toxins, unlike antibodies, and once isolated, the aptamers are reproduced with low cost and consistency by automated oligomer synthesis method. This was possible and economical.
- SELEX Systematic Evolution of Ligands by Exponential enrichment
- SELEX Systematic Evolution of Ligands by Exponential enrichment
- the present inventors have developed a microfluidic device employing the multiplex technique in PCT / US2009 / 054097, but the sequential separation of the aptamers bound to the target molecules is not possible at the same time. Only possible, there was a disadvantage that the means for separation of the aptamer is limited. This required the development of a new type of device and process suitable to shorten process run time and automate the entire process.
- each module After fabricating the fluidic device modules, each module can be connected to enable simultaneous multiplexing of aptamers for dozens or hundreds of target materials in dozens or hundreds of reaction chambers. It was confirmed that the connection can be automated and completed the present invention.
- Another object of the present invention is to provide a new method for screening aptamers, which is capable of screening a plurality of aptamers faster than before, and enables automation.
- the present invention provides a multiplex microfluidic device for nucleic acid aptamer selection comprising:
- the invention also provides a multiplex microfluidic device for aptamer screening comprising:
- connection region connecting the plurality of target molecule binding regions.
- the present invention also includes two or more of the multiplex microfluidic device as a unit, and the unit is connected to each other through a connection region, and provides a multiplex chip for aptamer selection.
- the invention also provides a high throughput screening method for nucleic acid aptamers comprising the following steps:
- step (d) collecting and amplifying the nucleic acids eluted in step (c);
- step (e) introducing the nucleic acids amplified in step (d) back into the binding channel of the multiplex microfluidic device and repeating steps (a) to (d),
- the present invention also provides a kit for high-speed sorting of aptamers containing the multiplex microfluidic device.
- the present invention also provides a method for analyzing nucleic acid released through the multiplex microfluidic device through a high speed sequencing method.
- FIG. 1 is a schematic diagram of the aptamer screening method according to the present invention.
- FIG. 2 is a schematic representation of a five-plex microfluidic device including a pneumatic valve opening and closing system.
- Figure 3 is a valve opening and closing system for controlling the switching between the binding channel and the elution channel to operate as a separate valve from the valve used for separating the binding channel between the plurality of target molecule binding region upon elution of the nucleic acid material reacted with the target
- Schematic diagram of a five-plex microfluidic device having "Pneumatic valve 1" is used to separate the binding channel between a plurality of target molecule binding regions upon elution of the nucleic acid material reacted with the target, and "Pneumatic valve 2" It is used to switch the operation between channels and dissolution channels.
- FIG. 4 is a photograph showing a flow chart of the device manufactured according to FIG. 2,
- (a) is a five-plex microfluidic device, and
- (b) is a main binding channel containing blue ink (aptamers to bind with target molecules)
- (C) is a photograph of introducing a washing solution to perform a washing process
- (d) is a photograph of injecting red ink (elution solution) into an elution channel
- e) In the valve-on state, the fluid is injected into the channel along the channel.
- (F) is a valve-on (flow-on) fluid flow in each elution channel.
- FIG. 6 is a photograph showing controlling the situation of FIG. 4 in accordance with an electrical signal flow.
- Figure 7 is an embodiment of a round 24 multiplex SELEX chip.
- 9 is an embodiment of a 24 Multiplex SELEX chip.
- 10 is an embodiment of a rounded 96 Multiplex SELEX chip.
- FIG. 11 is a schematic diagram of a microfluidic device in which a target coupling region (reaction chamber) is connected using a connecting region, that is, a tube.
- connection region 12 is a schematic view showing that the metal pin between the tube and the channel layer is used when connecting the target coupling region using the connection region, that is, the tube.
- Figure 13 is a structure showing that the adapter structure is used when connecting the target coupling region using a connection region, that is, a tube.
- FIG. 14A is a schematic diagram of a multiplex chip via module connection using a tube
- B is a photograph showing a practical example of module connection.
- 15 is a schematic diagram showing an example of performing an automated process of the method according to the invention.
- FIG. 16 illustrates the separation of aptamers for five targets of BSA (negative control), TBP, IIB, EGFP, and HSF using the apparatus of FIG. 2, and then Q- This is a graph confirmed by the PCR technique.
- nucleic acid refers to single- or double-stranded DNA, RNA and chemical modifications thereof, which modifications do not interfere with the amplification of selected nucleic acids, but are not limited to, but are not limited to, backbone. Modification, methylation, biphasic combination, substitution of 5-bromo-uracil, and the like.
- aptamer refers to a small single-stranded oligonucleotide that can specifically recognize a target with high affinity.
- target molecule refers to a macromolecular complex such as a protein or polypeptide, carbohydrate, lipid, pharmaceutical agent, low molecular material, organic non-pharmaceutical agent, and cell. It may be characterized in that it is selected from the group consisting of (macromolecular complex) and the like, but is not limited thereto.
- the low molecular weight substance includes nonpolar low molecular weight chemicals such as bisphenol A and the like.
- target molecule binding region refers to a reaction chamber in which a target molecule is bound by a sol-gel composition, beads, or the like, so that the aptamer may be bonded.
- microfluidic device refers to a device capable of controlling or manipulating the flow of microscopic microscopic solutions, such as ⁇ l, nL, pL, fL, and the like.
- the present invention relates to a multiplex microfluidic device for nucleic acid aptamer selection that can be applied to an improved Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process, such as FIG. 1.
- SELEX Systematic Evolution of Ligands by Exponential Enrichment
- the fluid channel connecting between the inlet and the outlet is a coupling channel, and each of the target molecular coupling regions is connected to the target.
- a fluid channel for eluting nucleic acid molecules bound to a target material from a molecular binding region is defined as an elution channel.
- a plurality of target molecule binding regions are formed in one coupling channel.
- the target molecules included in each target molecule combining region may be the same target molecules or two or more different target molecules.
- each target molecule is characterized by being a different target molecule.
- the target molecule binding region that is, a chamber in which the target molecule is combined with the introduced nucleic acid group, is connected to the other chamber through a binding channel, so that one end That is, when a nucleic acid population having a randomization sequence region is inserted at the inlet of the binding channel, the nucleic acid population enters each chamber and can bind to several target molecules at once. Therefore, the competition of aptamers can be induced for each target molecule, so that conditions for selecting aptamers having high affinity are satisfied.
- the coupling channel and the target molecular coupling region may be formed by a substrate cover deposited on the surface of the substrate, and the plurality of elution channels are also formed by a substrate lid deposited on the surface of the substrate. Can be formed.
- each single elution channel may have the same length, thereby making the volume and velocity of the elution solution constant.
- valve opening / closing system may function so that the nucleic acid that has been bound to the target molecule of the target molecule binding region may be eluted by the connected elution channel.
- the valve opening and closing system may automatically adjust the switching between the coupling channel and the elution channel in the multiplex microfluidic chip, and include two or more devices, that is, two or more valves, to allow each valve to operate differently.
- the valve opening and closing system may include a valve for regulating the flow of fluid in the binding channel and a valve for eluting a fluid including a nucleic acid bound to the target molecule into the eluting channel.
- This valve system regulates and optimizes the speed and flow of the fluid to reduce the pressure on the bonded portion of the substrate lid and chip, thereby minimizing fluid leakage.
- the valve opening and closing system may be any one of valves driven by heat, air pressure, thermo pneumatic pressure, hydraulic pressure, electrostatic force, electromagnetic force, magnetic force, phase change, and piezoelectric (pressure valve, frequency control valve, Micro-valve, directional coke, etc.), but when the nucleic acid pool having a randomization sequence region (target) binds to the target molecular binding reaction, the fluid containing the nucleic acid population is transferred through a binding channel.
- the present invention is not limited thereto as long as it is a valve opening and closing system that allows a fluid including the nucleic acid bound to the target molecule to flow into the elution channel.
- the pressure valve is characterized in that for changing the flow of the fluid by using a pressure, such as water pressure, air pressure, in the case of the micro-valve by artificially inserting a 2-way or 3-way valve of the fine size according to the artificial selection
- a pressure such as water pressure, air pressure
- the system using the directional coke is a directional active opening and closing system, which opens and closes horizontally during the integration reaction, that is, when performing the target molecule binding reaction of the nucleic acid, and allows fluid to flow through the binding channel and elutes the bound nucleic acid separately.
- it refers to a valve opening and closing system that enables eluting through the eluting channel vertically.
- a five-plex microfluidic device capable of sorting aptamers for five target molecules at once.
- the 5-plex microfluidic device (5-plex SELEX-on-a-Chip) according to the present invention has a bottom portion including a metal electrode as a heating means, and a lid forming a chamber, a channel, and a valve opening and closing system (pressure valve). It consists of parts (1 in Fig. 2).
- the bottom part which serves as a heater for eluting nucleic acid bound to the target molecule, was coated with PDMS (Polydimethylsiloxane) on a chip having five heater electrodes patterned with Cr / Au (2 in FIG. 2).
- the substrate cover has a first layer (3 in FIG. 2) having five chambers and a channel connecting the chambers thereon, and a second layer (4 in FIG. 2) forming a pneumatic valve thereon as the substrate cover thereon. ) And a third layer (5 of FIG. 2) serving as a support to allow fluid and air pressure to enter well. At this time, the substrate cover is all composed of PDMS.
- the diameter of the reaction chamber of the aptamer-target material was 1.8 mm, the height was 30 ⁇ m, and the channel width was 0.28 mm.
- the diameter of the chamber for applying air pressure was 0.5 mm, the height was 30 ⁇ m, and the width of the channel was 0.2 mm.
- surface treatment was performed with plasma under atmospheric pressure to make it very hydrophilic and used for bonding technology.
- the exposure time of Ar / O 2 for surface treatment was 2 seconds.
- An air pressure valve installed between the target molecular coupling regions, ie, a channel connecting the chamber to the chamber, creates an air pressure in the channel to prevent fluid from flowing from the chamber to the chamber and independently flows to each elution channel.
- the operation between the coupling channel and the elution channel is controlled automatically by a separate valve.
- each eluting channel for eluting the nucleic acid conjugate from the plurality of target molecule binding regions is the same, it was confirmed that the volume and the dissolution rate of the eluting channel are constant (FIG. 5).
- 'resistance' tilting of the channel
- Figure 6 shows the situation of the experiment using the pneumatic valve in terms of electromechanical signal flow (Signal flow).
- a mechanical input Mechanical input
- the air pressure (P) is guided for diaphragm deformation of the Vchamber Array, and this deformation ( ⁇ ) is It acts as a valve to block the flowing fluid.
- the multiplex microfluidic device for nucleic acid aptamer selection according to the present invention may be designed as shown in FIGS. 7 to 10.
- a 24 Multiplex SELEX chip which is designed in a round shape to simplify the shape of the chip. This can increase efficiency in the form of simultaneous elution like fractions.
- several chambers can be placed inside the round shape, allowing more SELEX. At this time, it is preferable to put the pump inside the chip to automatically perform the SELEX.
- FIG. 8 is also a 24 Multiplex SELEX chip, which can be manufactured about six on one mold, and thus, several chips can be manufactured at once and the efficiency can be increased during the experiment. At this time, by increasing the size of the chip, more than 24 SELEX chips can be formed, and since the chambers are independent shapes, there is an advantage that there is no influence on the sol-gel next to each other.
- FIG. 9 is also a 24 Multiplex SELES chip, which can also implement more than 24 SELEX chips if the size of the chip is increased.
- Figure 10 is a 96 Multiplex SELEX Chip, it is designed in a round (Disc-type) can be designed to simplify the shape of the chip, and to reduce the size of the chip. This allows for simultaneous elution like fractions, which increases efficiency when experimenting. In particular, several chambers can be placed inside the round shape, allowing more SELEX. At this time, it is preferable to put the pump inside the chip to automatically perform the SELEX.
- connection region connecting the plurality of target molecule binding regions.
- connection region is characterized in that one end can be separated from the target molecule binding region on one side or both sides, which may be characterized in that the tube as shown in FIG.
- the coupling channel and the target molecular coupling region are formed by a substrate lid (lid) deposited on the surface of the substrate, the tube also, the substrate lid (lid) deposited on the surface of the substrate It may be characterized in that it is connected to the hole (hole) of each target molecular coupling region formed by the "direct connection between the tube and the hole of the target molecular coupling region, the needle of the Hamilton syringe and the target molecular coupling region Direct connection between holes ", etc., but in this case, problems such as leakage of the reactants and non-reusability have occurred.
- the connection and separation of the inlet and the tube of the chamber can be freely and continuously used by connecting with a metal pin. All.
- an adapter (adapter) structure is characterized in that further deposited; can do.
- the adapter structure may be preferably characterized in that the plate-shaped structure made of PDMS (Polydimethylsiloxane).
- the target molecules in the target molecule-binding region can be fixed by various methods.
- a variety of substances can be fixed by using a sol-gel dispensing method.
- low-molecular substances such as chemicals and peptides, proteins, antibodies, and cells can be fixed in the reaction chamber in an active state without structural changes.
- a membrane filter may be used to prevent contamination of foreign substances or mixing of the target molecule-bead complexes between the target molecule binding regions, that is, the reaction chambers.
- the filter is additionally provided between the target molecule coupling region.
- Nucleic acid bound to the target molecule of the target molecule binding region can be eluted through the valve opening system through the elution channel, or eluted through one end of the connection region of the target molecule binding region, separation of the nucleic acid molecules bound to the target molecule Can be applied directly to the chip developed in various ways depending on the target molecule binding region, that is, the material fixed in the reaction chamber.
- high concentration salt treatment NaCl, KCl, MgCl 2 , Tris, etc.
- pH change HCl, NaOH treatment
- the nucleic acid can be isolated by inducing structural changes of the target protein.
- high concentration Imidazole and Glutathione treatment may induce competitive separation binding.
- high concentration chemical treatment causes nucleic acid molecule binding competition to be separated.
- the nucleic acid molecules bound to the target molecules were separated using heat.
- the aptamers form a structure and have a binding force to the target material, which is formed by hydrogen bonding between bases.
- the temperature at which DNA or RNA can be thermally denatured is called the melting temperature.
- the temperature for elution of aptamer is 65 ⁇ 95 °C.
- the existing separation method may be fused and applied together with heat generated in the chip.
- the apparatus according to the present invention may be such that the electrode as a heating means is provided with one electrode in close proximity to the plurality of target molecular coupling regions, or a plurality of electrodes are individually installed in proximity to each target molecular coupling region.
- the electrode as a heating means is provided with one electrode in close proximity to the plurality of target molecular coupling regions, or a plurality of electrodes are individually installed in proximity to each target molecular coupling region.
- the device according to the present invention has the advantage that the nucleic acid molecules can be separated at the same time by applying heat to the entire target molecule binding region as one electrode.
- the material of the metal electrode is not limited as long as it is for heat generation (eg Al, Cr / Au, etc.).
- this advantage is exponentially reduced time when silencing many target molecules at the same time as shown in FIGS.
- high-speed screening of more than 24-plex could not be realized due to inefficient dissolution time, but the multiple analysis such as 24-Felx and 96-Flex was made possible by improving the efficiency of high-speed screening by the opening and closing system of the valving system.
- the following points have been improved to prevent reactant leakage during valve operation and tube connection. That is, in the embodiment of the present invention by adjusting and optimizing the speed and flow of the fluid to reduce the pressure on the bonded portion of the substrate cover and the chip (chip) to minimize the leakage of the fluid.
- the PDMS Polydimethylsiloxane
- the substrate cover that forms the chamber and the channel is made of PDMS to form “All PDMS Chip”.
- the adhesion between the chip and the substrate cover is increased after plasma cleaning. To minimize fluid leakage.
- the substrate may be formed of any one of metals such as plastic, glass, silicon, and gold such as PDMS, PMMA (polymethylmethacrylate), and polystyrene.
- the substrate may be characterized by being coated with PDMS (Polydimethylsiloxane).
- the substrate cover is formed of polydimethylsiloxane (PDMS), thereby enabling the construction of an All PDMS Chip.
- PDMS Polydimethylsiloxane
- the substrate cover is made of PDMS and the thickness of the substrate cover is adjusted to reduce the back pressure generated in the channel when there is no fluid flow.
- the PDMS substrate cover may have a thickness of ⁇ 200 ⁇ m, more preferably 100 to 200 ⁇ m.
- the substrate cover is formed of polystyrene (polystyrene)
- the substrate may be formed of plastic, glass or silicon to manufacture a device such as plastic-polystyrene, glass-polystyrene, silicon-polystyrene.
- the multiplex microfluidic device can be used by connecting two or more by using it as a basic unit (module), and thus the present invention includes at least two of the multiplex microfluidic device as one unit,
- the unit relates to a multiplex chip for aptamer selection, characterized in that connected to each other through a connection region.
- a multiplex chip may be made by connecting basic units in series or parallel using a tube or a channel.
- a filter may be additionally installed in the connection region connecting the basic units to prevent foreign material contamination between the basic units.
- the present invention relates to a high throughput screening method for nucleic acid aptamers, wherein the multiplexed microfluidic device is used.
- the screening method according to the present invention may be characterized by including the following steps:
- step (d) collecting and amplifying the nucleic acids eluted in step (c);
- step (e) introducing the nucleic acids amplified in step (d) back into the binding channel of the multiplex microfluidic device and repeating steps (a) to (d),
- the method according to the present invention can be performed on 1 ⁇ 10 9 to 15 nucleic acid groups having different randomized alignment regions.
- the term randomized is used to describe segments of nucleic acid that, in principle, have any possible arrangement over a predetermined length. Randomization sequences have various lengths ranging from about 8 to 100 nucleotides or more as needed. The chemical or enzymatic reactions that result in random sequence segments may not produce computationally random sequences due to unknown bias or nucleotide preferences that may be present.
- the term randomization is used instead of random to reflect this possibility of bias from non-ideality. In currently known techniques such as sequential chemical synthesis, it is not known that large deviations occur. For short segments, the minimal bias that may be present is negligible.
- the method requires the initial preparation of the subject nucleic acid test mixture.
- Individual test nucleic acids may contain randomized regions flanking the configuration conserved in all nucleic acids in the mixture. conserveed regions are provided to facilitate amplification and selection of nucleic acids. Since there are many such known arrangements in the art, the selection of an arrangement is one that can be made by a person skilled in the art with a predetermined amplification method.
- the randomization region may have a fully or partial randomization arrangement.
- this portion of the nucleic acid may contain randomized fractions, with the fractions that are constantly fixed in all nucleic acids in the mixture.
- an alignment region known to bind to, or selected for binding to, a target protein may be integrated with the randomization region to improve binding or specificity of binding.
- the array diversity of the test mixture can be introduced or augmented by causing mutations in the nucleic acids of the test mixture during the screening / amplification process.
- the nucleic acid adopted in the test mixture can be of any length as it can be amplified.
- the method of the present invention is most practically employed in selecting from a number of array variants.
- the method is expected to be preferably employed to assess the binding of nucleic acid sequences of any reachable size from about four bases.
- the target molecules included in each target molecule binding region of the multiplex microfluidic device may be the same target molecules or two or more different target molecules.
- each target molecule is characterized by being a different target molecule.
- the target molecule binding region that is, a chamber in which the target molecule is combined with the introduced nucleic acid group, is connected to the other chamber through a binding channel, so that one end That is, when a nucleic acid population having a randomization sequence region is inserted at the inlet of the binding channel, the nucleic acid population enters each chamber and can bind to several target molecules at once. Therefore, the competition of aptamers can be induced for each target molecule, so that conditions for selecting aptamers having high affinity are satisfied.
- target molecules include macromolecule complexes such as proteins or polypeptides, carbohydrates, lipids, pharmaceutical agents, low molecular materials, organic non-pharmaceutical agents and cells. ) May be exemplified, but it is obvious that the molecule is the target of the aptamer.
- the nucleic acid group introduced into the device is brought into contact with the target molecule under conditions in which binding with the target molecule is preferred, and nucleic acids capable of specifically binding to the target molecule in the nucleic acid group are bound to the target molecule to be nucleic acid-targeted. Molecule pairs. Thus, subsequent removal of unbound nucleic acid is possible.
- amplification in the step of the present invention means a process or a combination of processes to increase the amount or number of copies or molecular types of molecules.
- the "amplification” process may be characterized by a PCR process, by using the cDNA copy production of the selected RNA, polymerase chain reaction using the increase the number of copies of each cDNA and the cDNA copy selected RNAs And obtaining an RNA molecule having the same configuration as. That is, as is known to those skilled in the art, any reaction or combination of reactions known in the art may be suitably employed, including methods such as direct DNA replication, direct RNA amplification, and the like.
- the amplification method should consequently represent essentially the proportion of the different arrangements in the initial mixture in terms of proportion of the amplified mixture.
- two or more of the multiplex microfluidic devices may be used as basic units (modules). That is, multiplex chips can be made by connecting basic units in series and in parallel using tubes or micro coupling channels. For example, it is possible to improve the 96-well plate type chip by connecting 24 4-plex chips.
- This modularization enables the automation of several tens to hundreds of well plate type chips, depending on the purpose of the experiment, which can be easily applied to sequencing (eg Solexa) and binding between materials (eg Octet).
- the repeating step of (e) is repeated until a predetermined selectivity is achieved, for example, the first experimental mixture, that is, a specific nucleic acid in the nucleic acid group having a randomized sequence region
- the repetition may continue until the degree of binding is achieved or until the minimum nucleic acid in the mixture is obtained.
- the repeating step may be performed within six times, and preferably, one to three times may be performed.
- sequencing was performed using high throughput sequencing of nucleic acids eluted from the multiplex microfluidic device in each repetition round of the method according to the present invention, followed by high speed sequencing in each repetition round.
- sequence analyzed by the nucleic acid sequence in which the relative ratio increases as the number of repetitions can be selected as an aptamer.
- the aptamer can be selected more quickly and simply.
- the bioinformatics analysis tool can be used to reduce the SELEX stage.
- the apparatus for performing the fast sequencing method may be directly performed by being connected to one side of each elution channel or each connection region of the multiplex microfluidic device.
- aptamers for the target material were selected and cloned into T-vectors to sequence the individual aptamers separately.
- the step (f) may be characterized in that to perform a target molecule affinity test on the finally eluted nucleic acid to select a nucleic acid confirmed affinity as an aptamer.
- the target molecule affinity test may be characterized as a high-speed affinity test method, for example, may use the Octet system as a fast affinity test, the Octet system using a bio-layer interferometry (BLI) technology Label-free kinetic analysis and quantitation for the analysis of binding between substances.
- BBI bio-layer interferometry
- BLI Bio-Layer Interferometry
- This thickness change is represented by a change in the wave pattern of the white light reflected through the sensor surface to prevent the coupling between materials. It is a technique to measure. It can be multiplexed by kinetic screening or affinity testing of 96 samples within two hours, and can measure interactions between proteins as well as peptides or small molecule materials.
- the microfluidic device according to the present invention may be provided in a 96well plate type based on a basic unit, and then connected to the Octet system.
- the method according to the invention is characterized by being able to automate the performance of each step, as shown in FIG. 15.
- the conventional SELEX process is an intensive collection of bio-related experiments, and the success or failure of the experiment was determined by the experimenter's ability and know-how. Therefore, the need for standardization and automation was highly demanded.
- the device according to the present invention is a SELEX-on-chip, and can be designed according to the generalized 96 / 384-well plate standard to enable automation of the entire SELEX process in connection with existing equipment.
- the device according to the present invention that is, SELEX-on-a-Chip is composed of a single unit (module) type, by connecting the basic unit in series and parallel by using a tube multiplex chip (eg, 96-well type, 384-well type, etc.) and physical connection using standardized SELEX chips and tubes or chamber opening / closing operation by a valve system to automatically control the separation and recovery of aptamers into 96-well plates. Therefore, it is easy to apply to the existing 96 / 384-well plate-based analyzer (PCR, high throughput sequencer, affinity measuring device).
- PCR high throughput sequencer
- affinity measuring device affinity measuring device.
- the aptamer targeting 101apt as an aptamer for five types of targets of BSA (negative control), TBP, IIB, EGFP, and HSF using the 5-plex microfluidic device of FIG. 2.
- BSA negative control
- TBP TBP
- IIB TBP
- EGFP EGFP
- HSF HSFapt
- the method according to the invention may be provided in the form of a kit, in order to increase portability. That is, in another aspect, the present invention relates to a kit for high-speed sorting of aptamers containing the multiplex microfluidic device.
- the kit may additionally provide a reagent for amplification and sequencing of the eluted nucleic acid in a separate container or may be provided in a separate reaction part.
- the high-speed screening kit of aptamers may take the form of bottles, tubs, small sachets, envelopes, tubes, ampoules, etc., which may be partially or wholly plastic, glass, paper, etc. , Foils, waxes and the like.
- the container may be equipped with a fully or partially separable stopper, which may initially be part of the container or attached to the container by mechanical, adhesive, or other means.
- the container may also be equipped with a stopper, which may be accessible to the contents by a needle.
- the kit may include an external package, which may include instructions for use of the components.
- the present invention has an effect of providing a multiplex microfluidic device for nucleic acid aptamer selection and a high speed screening method of nucleic acid aptamer using the same.
- the multiplex fluid apparatus according to the present invention not only can detect each aptamer for a plurality of targets at the same time, but also has the advantage of greatly reducing the throughput and the processing time as compared to the conventional multiplex technique. It is very useful to perform the aptamer sorting process in conjunction with the high speed sequencing method to greatly reduce the target binding / separation / amplification process round and to perform the automated process.
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Abstract
Description
Claims (54)
- 다음을 포함하는 핵산 압타머 선별을 위한 멀티플렉스 미세 유체 장치:(a) 입구 및 출구 사이를 연결하는 결합 채널을 포함하는 기판;(b) 상기 결합 채널 내에 형성되는 다수개의 타겟분자결합영역;(c) 상기 다수개의 타겟분자결합영역에 각각 연결되는 다수개의 용출채널; 및(d) 밸브개폐시스템.
- 제1항에 있어서, 상기 결합 채널 및 타겟분자결합영역은 상기 기판의 표면에 증착되는 기판 덮개에 의하여 형성되는 것을 특징으로 하는 장치.
- 제1항에 있어서, 상기 다수개의 용출채널은 상기 기판의 표면에 증착되는 기판 덮개(lid)에 의하여 형성되는 것을 특징으로 하는 장치.
- 제1항에 있어서, 상기 밸브개폐시스템은 결합 반응 시 상기 결합 채널을 통하여 상기 다수개의 타겟분자결합영역 사이로 유체가 흐르게 하고, 결합이 종료된 후에는 결합 채널을 통하여 상기 다수개의 타겟분자결합영역 간 유체가 흐르지 않고 각 타겟분자결합영역에 연결된 용출채널로 타겟분자에 결합된 핵산을 포함하는 유체가 용출될 수 있도록 하는 것을 특징으로 하는 장치.
- 제4항에 있어서, 상기 밸브개폐시스템은 결합채널 내의 유체 흐름을 조절하는 밸브와 상기 타겟분자에 결합된 핵산을 포함하는 유체를 용출채널로 용출시키는 밸브를 포함하는 것을 특징으로 하는 장치.
- 제4항에 있어서, 상기 밸브개폐시스템은 열, 공기압, 열공압, 유압, 정전기력, 전자력, 자력, 상변화 및 압전 중 어느 하나를 밸브개폐를 위한 구동요소로 하는 시스템인 것을 특징으로 하는 장치.
- 제1항에 있어서, 상기 용출채널로 용출되는 용출용액의 부피 및 용출 속도가 일정하도록 상기 다수개의 용출채널의 길이를 서로 동일하게 하는 것을 특징으로 하는 장치.
- 제1항에 있어서, 상기 각 타겟분자결합영역의 타겟분자와 결합된 핵산을 각 타겟분자결합영역에 연결된 각 용출채널로 용출되도록 하는 용출수단을 추가로 포함하는 것을 특징으로 하는 장치.
- 제8항에 있어서, 상기 용출수단은 상기 결합 채널의 다수개의 타겟분자결합영역에 근접하여 설치되는 가열수단인 것을 특징으로 하는 장치.
- 제9항에 있어서, 상기 가열수단은 전극인 것을 특징으로 하는 장치.
- 제10항에 있어서, 하나의 전극이 상기 다수개의 타겟분자결합영역에 근접하여 설치되는 것을 특징으로 하는 장치.
- 제10항에 있어서, 다수개의 전극이 각 타겟분자결합영역에 근접하여 각각 개별적으로 설치되는 것을 특징으로 하는 장치.
- 제2항에 있어서, 상기 기판 덮개는 PDMS(Polydimethylsiloxane)로 형성되는 것을 특징으로 하는 장치.
- 제13항에 있어서, 상기 PDMS 기판덮개의 두께를 100~200㎛로 하는 것을 특징으로 하는 장치.
- 제1항에 있어서, 기판은 플라스틱, 유리, 실리콘 및 금속 중 어느 하나로 형성되는 것을 특징으로 하는 장치.
- 제15항에 있어서, 상기 플라스틱은 PDMS, PMMA (Polymethylmethacrylate) 및 폴리스티렌(polystyrene) 중 어느 하나로 형성되는 것을 특징으로 하는 장치.
- 제15항에 있어서, 상기 기판은 PDMS (Polydimethylsiloxane), PVA(Polyvinyl alcohol) 및 폴리비닐 아세테이트(polyvinyl acetate) 중 어느 하나로 코팅한 것임을 특징으로 하는 장치.
- 제2항에 있어서, 상기 기판 덮개는 폴리스티렌(polystyrene)으로 형성되고, 기판은 플라스틱, 유리 및 실리콘 중 어느 하나로 형성되는 것을 특징으로 하는 장치.
- 제1항에 있어서, 상기 타겟분자결합영역 사이에 추가로 필터가 설치되는 것을 특징으로 하는 장치.
- 다음을 포함하는 압타머 선별을 위한 멀티플렉스 미세 유체 장치:(a) 입구 및 출구 사이를 연결하는 결합 채널을 포함하는 기판;(b) 상기 결합 채널 내에 형성되는 다수개의 타겟분자결합영역; 및(c) 상기 다수개의 타겟분자결합영역을 연결하는 연결영역.
- 제20항에 있어서, 상기 연결영역은 일측 또는 양측의 타겟분자결합영역으로부터 일 말단이 분리될 수 있는 것을 특징으로 하는 장치.
- 제20항에 있어서, 상기 연결영역은 튜브인 것을 특징으로 하는 장치.
- 제20항에 있어서, 상기 결합 채널 및 타겟분자결합영역은 상기 기판의 표면에 증착되는 기판 덮개(lid)에 의하여 형성되는 것을 특징으로 하는 장치.
- 제22항에 있어서, 상기 튜브는 상기 기판의 표면에 증착되는 기판 덮개(lid)에 형성되어 있는 각 타겟분자결합영역의 홀(hole)로 연결되는 것을 특징으로 하는 장치.
- 제24항에 있어서, 상기 튜브는 금속핀을 통하여 상기 타겟분자결합영역의 홀로 연결되는 것을 특징으로 하는 장치.
- 제24항에 있어서, 상기 튜브와 타겟분자결합영역의 홀 사이에 어뎁터 구조물이 추가로 증착된 것을 특징으로 하는 장치.
- 제20항에 있어서, 상기 각 타겟분자결합영역의 타겟분자와 결합된 핵산을 각 타겟분자결합영역에 연결된 각 연결영역의 일측으로 용출되도록 하는 용출수단을 추가로 포함하는 것을 특징으로 하는 장치.
- 제27항에 있어서, 상기 용출수단은 상기 결합 채널의 다수개의 타겟분자결합영역에 근접하여 설치되는 가열 수단인 것을 특징으로 하는 장치.
- 제28항에 있어서, 상기 가열수단은 전극인 것을 특징으로 하는 장치.
- 제29항에 있어서, 하나의 전극이 상기 다수개의 타겟분자결합영역에 근접하여 설치되는 것을 특징으로 하는 장치.
- 제29항에 있어서, 다수개의 전극이 각 타겟분자결합영역에 근접하여 각각 개별적으로 설치되는 것을 특징으로 하는 장치.
- 제23항에 있어서, 상기 기판 덮개는 PDMS (Polydimethylsiloxane)로 형성되는 것을 특징으로 하는 장치.
- 제32항에 있어서, 상기 PDMS 기판덮개의 두께를 100~200㎛로 하는 것을 특징으로 하는 장치.
- 제20항에 있어서, 기판은 플라스틱, 유리, 실리콘 및 금속 중 어느 하나로 형성되는 것을 특징으로 하는 장치.
- 제34항에 있어서, 상기 기판은 PDMS (Polydimethylsiloxane), PVA(Polyvinyl alcohol) 및 폴리비닐 아세테이트(polyvinyl acetate) 중 어느 하나로 코팅한 것임을 특징으로 하는 장치.
- 제23항에 있어서, 상기 기판 덮개는 폴리스티렌(polystyrene)으로 형성되고, 기판은 플라스틱, 유리 및 실리콘 중 어느 하나로 형성되는 것을 특징으로 하는 장치.
- 제20항에 있어서, 상기 타겟분자결합영역 사이에 추가로 필터가 설치되는 것을 특징으로 하는 장치.
- 제1항 내지 제37항의 멀티플렉스 미세 유체 장치를 일 단위체로 하여 둘 이상 포함하되, 상기 단위체는 서로 연결영역을 통하여 연결된 것임을 특징으로 하는, 압타머 선별을 위한 멀티플렉스 칩.
- 제38항에 있어서, 상기 단위체 간 연결영역은 튜브 또는 채널인 것을 특징으로 하는 멀티플렉스 칩.
- 제38항에 있어서, 상기 단위체 간 연결영역에 추가로 필터가 설치되는 것을 특징으로 하는 멀티플렉스 칩.
- 다음 단계를 포함하는 핵산 압타머의 고속(high Throughput) 선별방법:(a) 랜덤화 배열 영역을 가지는 단일가닥 핵산들로 구성되는 핵산 집단을 제1항 내지 제37항 중 어느 한 항의 멀티플렉스 미세 유체 장치의 결합 채널로 도입하여 결합 채널 내의 타겟분자결합영역의 타겟분자와 반응시키는 단계;(b) 상기 타겟분자와 결합하지 않은 핵산들을 상기 멀티플렉스 미세 유체 장치로부터 제거하는 단계;(c) 상기 타겟분자와 결합된 핵산들을 상기 멀티플렉스 미세 유체 장치의 각 용출채널 또는 각 연결영역의 일측으로 용출하는 단계;(d) 상기 (c) 단계에서 용출된 핵산들을 수집하여 증폭시키는 단계;(e) 상기 (d) 단계에서 증폭된 핵산들을 다시 상기 멀티플렉스 미세 유체 장치의 결합 채널로 도입시켜 상기 (a) 내지 (d) 단계를 반복하는 단계,이때, 최종 반복라운드에서는 용출된 핵산의 증폭과정을 수행하지 않는 것을 특징으로 함; 및(f) 최종 반복라운드에서 용출된 핵산을 압타머로서 선별하는 단계.
- 제41항에 있어서, 상기 핵산 집단은 서로 다른 랜덤화 배열 영역을 가지는 1×109~15개의 핵산으로 구성되는 것을 특징으로 하는 방법.
- 제41항에 있어서, 상기 멀티플렉스 미세 유체 장치는 서로 상이한 타겟분자를 포함하는 타겟분자결합영역들을 포함하는 것을 특징으로 하는 방법.
- 제41항에 있어서, 상기 멀티플렉스 미세 유체 장치는 서로 동일한 타겟분자를 포함하는 타겟분자결합영역들을 포함하는 것을 특징으로 하는 방법.
- 제41항에 있어서, 상기 타겟분자는 단백질 또는 폴리펩타이드, 탄수화물(carbohydrate), 지질(lipid), 약학제제(pharmaceutical agent), 저분자물질, 유기성 비약학제제(organic non-pharmaceutical agent) 및 거대분자복합체 (macromolecular complex)로 구성된 군에서 선택된 것임을 특징으로 하는 방법.
- 제41항에 있어서, 상기 멀티플렉스 미세 유체 장치가 둘 이상 연결되어 있는 것을 특징으로 하는 방법.
- 제46항에 있어서, 상기 멀티플렉스 미세 유체 장치는 튜브 또는 채널에 의하여 연결되는 것을 특징으로 하는 방법.
- 제41항에 있어서, 각 반복라운드에서 상기 멀티플렉스 미세 유체 장치로부터 용출된 핵산들에 대하여 고속 시퀀싱법(high throughput sequencing)을 이용하여 서열분석을 수행한 다음, 각 반복라운드에서 고속 시퀀싱법에 의하여 분석된 서열을 비교하여 반복횟수의 증가에 따라 상대적인 비율이 증가하는 핵산서열을 압타머로서 선별하는 것을 특징으로 하는 방법.
- 제41항에 있어서, 상기 (e)단계의 반복은 1 내지 6회 수행되는 것을 특징으로 하는 방법.
- 제48항에 있어서, 상기 고속 시퀀싱법을 수행하는 장치는 상기 멀티플렉스 미세 유체 장치의 각 용출채널 또는 각 연결영역의 일측과 연결된 것을 특징으로 하는 방법.
- 제41항에 있어서, 상기 (f) 단계는 최종적으로 용출된 핵산에 대하여 추가로 타겟분자 친화성 테스트를 수행하여 친화도가 확인된 핵산을 압타머로서 선별하는 것을 특징으로 하는 방법.
- 제51항에 있어서, 상기 타겟분자 친화성 테스트는 고속 친화성 테스트 방법인 것을 특징으로 하는 방법.
- 제41항에 있어서, 각 단계의 수행은 자동화되어 있는 것을 특징으로 하는 방법.
- 제1항 내지 제37항 중 어느 한 항의 멀티플렉스 미세 유체 장치를 함유하는 압타머의 고속 선별용 키트.
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CN201180004708.9A CN102639720B (zh) | 2010-10-05 | 2011-10-05 | 用于筛选核酸适配子的多元微流体装置及利用此装置的核酸适配子的高通量筛选方法 |
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WO2013019714A1 (en) | 2011-07-29 | 2013-02-07 | The Trustees Of Columbia University In The City Of New York | Mems affinity sensor for continuous monitoring of analytes |
DE102011085473A1 (de) * | 2011-10-28 | 2013-05-02 | Albert-Ludwigs-Universität Freiburg | Verfahren zur Identifikation von Aptameren |
CN103278643B (zh) * | 2013-05-16 | 2015-05-27 | 中国科学院上海微系统与信息技术研究所 | 一种用于微量蛋白检测的微芯片的制备方法 |
WO2016022696A1 (en) | 2014-08-05 | 2016-02-11 | The Trustees Of Columbia University In The City Of New York | Method of isolating aptamers for minimal residual disease detection |
KR101849557B1 (ko) * | 2014-09-29 | 2018-04-17 | 동국대학교 산학협력단 | 신규 세포 타겟 압타머 제조방법 |
WO2016059619A2 (en) * | 2014-10-17 | 2016-04-21 | Ecole Polytechnique Federale De Lausanne (Epfl) | Microfluidic device and method for isolation of nucleic acids |
US10546650B2 (en) * | 2015-10-23 | 2020-01-28 | Google Llc | Neural network for processing aptamer data |
US10882045B2 (en) * | 2016-01-08 | 2021-01-05 | Hewlett-Packard Development Company, L.P. | Polymerase chain reaction device including ejection nozzles |
CN109694806B (zh) * | 2017-10-24 | 2021-11-19 | 湖南大学 | 一种微流体装置及基于微流体装置的单细胞核酸适体单轮筛选方法 |
TWI675106B (zh) * | 2018-03-21 | 2019-10-21 | 緯創資通股份有限公司 | 液體處理模組、液體檢測系統及利用液體處理模組進行檢測的檢測方法 |
CN110564815A (zh) * | 2019-09-03 | 2019-12-13 | 集美大学 | 一种筛选获得极短序列核酸适配体的方法 |
KR102414945B1 (ko) | 2019-10-02 | 2022-07-05 | 한국식품연구원 | 핵산 선별용 졸-겔 나노 필터 및 이를 이용한 핵산 선별 방법 |
BR102020004436A8 (pt) | 2020-03-05 | 2022-11-22 | Bioptamers Inc | Dispositivo microfluídico |
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