WO2022260463A1 - Diagnostic kit and diagnostic method - Google Patents
Diagnostic kit and diagnostic method Download PDFInfo
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- WO2022260463A1 WO2022260463A1 PCT/KR2022/008177 KR2022008177W WO2022260463A1 WO 2022260463 A1 WO2022260463 A1 WO 2022260463A1 KR 2022008177 W KR2022008177 W KR 2022008177W WO 2022260463 A1 WO2022260463 A1 WO 2022260463A1
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- aptamer
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Classifications
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- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- 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
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/548—Carbohydrates, e.g. dextran
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- G—PHYSICS
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/552—Glass or silica
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- 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
- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
- C12Q2525/10—Modifications characterised by
- C12Q2525/205—Aptamer
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- 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
- C12Q2531/00—Reactions of nucleic acids characterised by
- C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
- C12Q2531/113—PCR
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- 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
- C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
- C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
Definitions
- the present invention relates to an aptamer and a diagnostic kit including the same, and in particular, a target substance and a primary aptamer that specifically binds to the target substance are formed by binding to a primary aptamer-target substance complex and specific binding It is about a secondary aptamer that does.
- Test methods are largely classified into PCR tests to check for virus infection through nucleic acid amplification after collecting biological samples, antibody tests to check whether antibodies are formed, and antigen tests to directly detect viruses, and active research is continuing in each field.
- Aptamers are nucleic acid materials composed of DNA or RNA, and can specifically bind to specific substances such as proteins, cells, and microorganisms. Aptamers are short-length nucleic acid sequences that are superior in stability to antibodies, enabling long-term preservation and having high affinity for a target, and thus are in the spotlight for application in various technical fields. Due to the excellence of these aptamers, there is a movement to apply aptamers to infectious disease testing methods, and a pair of aptamers that bind to two parts of the virus is bound to the virus and detects whether the virus is detected through a marker that labels it, such as a fluorescent substance. method is known.
- Chinese Patent Publication No. 106443003 discloses that an aptamer labeled with a fluorescent receptor is attached to a conjugation pad, and a probe labeled with a fluorescent material is attached to a detection line so that a FRET phenomenon occurs between them depending on the presence or absence of a target material. A fluorescence quenching test strip is presented.
- Korean Patent Publication No. 106443003 discloses that an aptamer labeled with a fluorescent receptor is attached to a conjugation pad, and a probe labeled with a fluorescent material is attached to a detection line so that a FRET phenomenon occurs between them depending on the presence or absence of a target material. A fluorescence quenching test strip is presented.
- 2017-0103119 discloses that a DNA aptamer complex intercalated with a fluorescent dye is bound to a complementary strand fixed to a support to form a double helix, and when a target substance is present, the DNA aptamer complex A method for visualizing and detecting fluorescence staining emitted as it binds to and detaches from a target material is proposed.
- the aptamer of the present invention was devised to solve the above problems, and specifically binds to the target substance and the primary aptamer-target substance complex formed by binding the target substance and the primary aptamer that binds specifically to the target substance. Its purpose is to provide an aptamer, which is a secondary aptamer.
- the present invention is a primary aptamer formed by combining a target substance and a primary aptamer that specifically binds to the target substance - a secondary aptamer that specifically binds to the target substance complex;
- Another object is to provide an aptamer complex comprising a; and a label that binds to and labels the secondary aptamer.
- the present invention provides a diagnostic kit including a target substance and a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding of a primary aptamer that specifically binds to the target substance. that serves another purpose.
- the present invention provides a target substance detection method using a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding a target substance and a primary aptamer that specifically binds to the target substance to do for another purpose.
- the present invention may also have as its object to achieve other objects that can be easily derived by a person of ordinary skill in the art from these objects and the overall description of this specification in addition to the above clear objects.
- the aptamer of the present invention is formed by combining a target substance and a primary aptamer that specifically binds to the target substance, and a secondary aptamer-target substance complex that specifically binds to the target substance. It is characterized in that it is an aptamer.
- the target substance is an antigen, antibody, virus, oligonucleotide, protein, carbohydrate, fructose, glycosaccharide, oligosaccharide, hormone, receptor, peptide, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye , nutrients, growth factors, tissues, inorganic organic nutrients, harmful environmental substances, or oligosaccharides of glycoproteins.
- the secondary aptamer can recognize a three-dimensional structure formed by binding the primary aptamer and the target material.
- the secondary aptamer can specifically bind to the target material.
- the binding affinity of the secondary aptamer to the primary aptamer-target material complex may be 2 to 2000 times, 2 to 1800 times, or 2 to 1500 times the binding affinity to the target material.
- the aptamer complex according to the present invention includes a primary aptamer formed by binding a target substance and a primary aptamer specifically binding to the target substance, and a secondary aptamer specifically binding to the target substance complex; and a label that binds to and labels the secondary aptamer.
- the marker may be selected from the group consisting of a fluorescent substance, a luminous substance, a quantum dot, a quantum dot bead, gold nanoparticles, europium, a dye, a radioactive label, an electrochemical functional group, an enzyme, an enzyme substrate, and mixtures thereof.
- the marker is cadmium sulfide (CdS), cadmium selenide (CdSe), cadmium tellenide (CdTe), zinc sulfide (ZnS), zinc selenide (ZnSe), zinc tellenide (ZnTe), zinc oxide (ZnO ), zinc oxide zinc selenide (ZnOZnSe), zinc oxide zinc sulfide (ZnOZnS), cadmium oxide (CdO), cadmium selenium sulfide (CdSeS), cadmium selenium tellenide (CdSeTe), cadmium sulfide tellenide (CdSTe), cadmium zinc Sulfide (CdZnS), Cadmium Zinc Selenide (CdZnSe), Cadmium Zinc Tellenide (CdZnTe), Zinc Selenium Sulfide (ZnSeS), Zinc Selenium Tellenide (ZnS
- the marker may form a bond with the secondary aptamer through a covalent bond, an ionic bond, or an electrostatic bond.
- the marker may form a bond with the secondary aptamer through a bond using an EDC or EDC / NHS (or sulfo-NHS) reaction, a disulfide bond (S-S), or a bond using maleimide.
- the diagnostic kit according to the present invention includes a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding a target substance and a primary aptamer that specifically binds to the target substance. characterized by
- the diagnostic kit may be a lateral flow diagnostic strip (LFA), an enzyme immunoassay kit (ELISA), or a biochip.
- LFA lateral flow diagnostic strip
- ELISA enzyme immunoassay kit
- biochip a biochip
- the diagnostic kit may include a primary aptamer that specifically binds to a target substance.
- At least one of the primary aptamer and the secondary aptamer may be immobilized to the support.
- the primary aptamer and the secondary aptamer may be non-fixed.
- the method for detecting a target substance uses a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding a target substance and a primary aptamer that specifically binds to the target substance. characterized by use.
- the primary aptamer or the secondary aptamer may be labeled with a signal generating substance.
- the primary aptamer or secondary aptamer may be labeled with a substance selected from the group consisting of a fluorescent substance, a luminescent substance, a staining reagent, an enzyme dye (HRP), and a mixture thereof.
- the target material detection method may include contacting a sample suspected of having a target material with the primary aptamer and the secondary aptamer simultaneously or with a time difference.
- At least one of the primary aptamer and the secondary aptamer may be immobilized to the support.
- the target material detection method may include contacting a sample suspected of having a target material with a non-fixed primary aptamer and a non-fixed secondary aptamer at the same time or with a time difference; and immobilizing at least one of the primary aptamer and the secondary aptamer to the support.
- At least one of the primary aptamer and the secondary aptamer may have a functional group capable of binding to the support.
- the target substance detection method determines whether the target substance in the sample is formed according to whether a first aptamer-target substance-second aptamer complex formed by binding the first aptamer-target substance complex and the second aptamer is formed. It may include a step of determining existence or nonexistence.
- first aptamer-target substance-secondary aptamer complex is formed can be determined through PCR.
- first aptamer-target substance-secondary aptamer complex is formed can be determined through a visual signal.
- the aptamer and the target substance detection method using the same according to the present invention have excellent detection sensitivity, simple operation, and do not require labeling of a secondary reagent capable of binding to the target substance.
- an aptamer instead of an antibody small molecule detection can be facilitated and specificity can be increased.
- FIG. 1 is a schematic diagram of an aptamer according to an embodiment of the present invention.
- FIG. 2 is a schematic diagram of an assay for confirming non-specific binding between a primary aptamer and a secondary aptamer according to an embodiment of the present invention.
- FIG. 3 is a test result for confirming non-specific binding between a primary aptamer and a secondary aptamer according to an embodiment of the present invention.
- FIG. 4 is a schematic diagram of a process for binding a secondary aptamer to a primary aptamer-target material complex according to an embodiment of the present invention.
- FIG. 5 is a binding affinity test result of a secondary aptamer to a primary aptamer-target material complex according to an embodiment of the present invention.
- FIG. 6 is a schematic diagram of a sandwich method assay of a primary aptamer and a secondary aptamer according to an embodiment of the present invention.
- FIG. 7 is a result of a sandwich method assay of a primary aptamer and a secondary aptamer according to an embodiment of the present invention.
- FIG. 8 is a schematic diagram of a method for screening secondary aptamers according to an embodiment of the present invention.
- FIG. 10 shows the structure and operation of a lateral flow sensor, which is a diagnostic kit according to an embodiment of the present invention.
- 11 is a lateral flow sensor test result for selecting a primary aptamer-secondary aptamer pair according to an embodiment of the present invention.
- FIG. 13 is a fluorescence reader measurement result according to a target concentration of a diagnostic kit according to an embodiment of the present invention.
- first and second may be used to describe various components, but the components should not be limited by the terms. These terms are only used for the purpose of distinguishing one component from another. For example, a first element may be termed a second element, and similarly, a second element may be termed a first element, without departing from the scope of the present invention.
- Aptamer is a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that specifically binds to a target substance. It is easy to respond to variants such as
- the aptamer of the present invention is characterized in that it is a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding a target substance and a primary aptamer that specifically binds to the target substance.
- the target substance is an antigen, antibody, virus, oligonucleotide, protein, carbohydrate, fructose, glycosaccharide, oligosaccharide, hormone, receptor, peptide, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient , growth factors, tissues, inorganic organic nutrients, harmful environmental substances, or oligosaccharides of glycoproteins.
- the primary aptamer is an aptamer that specifically binds to a target substance.
- the secondary aptamer may not specifically bind to a single target substance.
- the secondary aptamer also has binding affinity to the target substance or can bind specifically to the target substance like the primary aptamer, but in this case, the secondary aptamer has binding affinity for the primary aptamer-target substance complex It can be characterized as significantly higher than the binding affinity for the target substance. This may mean that the target of the secondary aptamer is not a simple target substance but a primary aptamer-target substance complex.
- one aptamer recognizes a conjugate of another aptamer and a target material, so specificity and sensitivity can be greatly improved.
- the secondary aptamer can be prepared as follows. First, a primary aptamer is selected for a specific target material through the selex method. At this time, a known primary aptamer that specifically binds to the target material may be used. After binding the selected 1st aptamer to the beads immobilized with the target substance, a selex round is run on this conjugate through the aptamer library, and then the 2nd aptamer is selected through the process of finding the most optimal pair through kd assay do. Therefore, the secondary aptamer of the present invention is clearly distinguished from the primary aptamer selected by immobilizing a simple target material in terms of target, base sequence, and three-dimensional structure.
- the secondary aptamer can be prepared as follows with reference to FIG. A primary aptamer is immobilized on a surface (plate, bead, etc.), and a binding reaction is induced to a random library having a random base sequence in a state in which a target material-primary aptamer complex is formed. Thereafter, those that do not bind are removed by washing, and finally, the aptamer that binds to the primary aptamer and the target complex is finally selected.
- the secondary aptamer can be prepared as follows with reference to FIG. Biotin-labeled primary aptamers selected by the general SELEX method are immobilized on a streptavidin-coated plate. Thereafter, non-binding materials are removed by washing, and the target is combined with the primary aptamer to form a primary aptamer-target complex. Then, in order to select the secondary aptamer, the random library is treated in a plate well (plate No.
- FIG. 1 is a schematic diagram of the binding principle of a secondary aptamer according to an embodiment of the present invention.
- the left side of the arrow is a target substance-primary aptamer complex in which the target substance 30 and the primary aptamer 10 are bound, and the secondary aptamer 20 with very high affinity specifically binds to this complex.
- a complex can be formed as shown to the right of the arrow.
- the secondary aptamer can recognize a three-dimensional structure formed by binding the primary aptamer and the target material.
- the secondary aptamer may simultaneously form a bond with at least a portion of the primary aptamer and at least a portion of the target material.
- the secondary aptamer may form a bond with at least one of the structure of the primary aptamer formed by binding the target material and the primary aptamer or the structure of the target material.
- the binding affinity of the secondary aptamer to each of them may be low or low.
- the secondary aptamer may not specifically or non-specifically bind with the primary aptamer.
- the secondary aptamer has a binding affinity for the primary aptamer-target material complex that is 2-fold, 5-fold, 10-fold, 20-fold, or 50-fold higher than the binding affinity for the target material. , 100 times or more, 1000 times or more, or 10000 times or less, 5000 times or less, 2000 times or less, 1000 times or less, 100 times or less, 50 times or less, specifically 2 to 2000 times, 2 to 1800 times, or 2 to 1500 times, 2 to 1000 times, 100 to 1000 times, 2 to 100 times, 10 to 100 times, 2 to 10 times, or 2 to 5 times.
- the binding affinity can be determined by labeling the end of the secondary aptamer with a substance capable of generating a measurable signal and then comparing the intensity of the signal. For example, binding affinity can be compared by labeling the end of the secondary aptamer with biotin, HRP, etc., and then measuring and comparing fluorescence values.
- the aptamer complex according to the present invention includes a primary aptamer formed by binding a target substance and a primary aptamer specifically binding to the target substance, and a secondary aptamer specifically binding to the target substance complex; and a label that binds to and labels the secondary aptamer.
- the marker may be selected from the group consisting of a fluorescent substance, a luminous substance, a quantum dot, a quantum dot bead, gold nanoparticles, europium, a dye, a radioactive label, an electrochemical functional group, an enzyme, an enzyme substrate, and mixtures thereof.
- the label is a quantum dot
- the quantum dot may be a group 12-16 compound or a group 13-15 compound, and may include a semiconductor compound capable of forming quantum dots.
- the quantum dots may have a core-shell structure.
- the quantum dots are cadmium sulfide (CdS), cadmium selenide (CdSe), cadmium tellenide (CdTe), zinc sulfide (ZnS), zinc selenide (ZnSe), zinc tellenide (ZnTe), zinc oxide (ZnO), zinc Zinc oxide selenide (ZnOZnSe), zinc oxide zinc sulfide (ZnOZnS), cadmium oxide (CdO), cadmium selenium sulfide (CdSeS), cadmium selenium tellenide (CdSeTe), cadmium sulfide tellenide (CdSTe), cadmium zinc sulfide (CdZn
- the marker may form a bond with the secondary aptamer through a covalent bond, an ionic bond, or an electrostatic bond.
- the marker may form a bond with the secondary aptamer through a bond using an EDC or EDC / NHS (or sulfo-NHS) reaction, a disulfide bond (S-S), or a bond using maleimide.
- the diagnostic kit according to the present invention includes a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding a target substance and a primary aptamer that specifically binds to the target substance. characterized by
- the diagnostic kit may be a diagnostic kit using specific binding, and may be a known detection device for detecting a target substance, such as a lateral flow diagnostic strip (LFA), an enzyme immunoassay kit (ELISA), or a biochip.
- LFA lateral flow diagnostic strip
- ELISA enzyme immunoassay kit
- biochip a biochip
- the diagnostic kit may include a primary aptamer that specifically binds to a target substance.
- At least one of the primary aptamer and the secondary aptamer may be immobilized to the support.
- the support may mean forming the inner surface of the diagnostic kit.
- the primary aptamer and the secondary aptamer may be non-fixed. This may mean a state in which they can freely move within the diagnostic kit. However, in the target material detection process, a step of immobilizing the non-immobilized primary aptamer or the secondary aptamer may be performed.
- the lateral flow sensor includes a sample input pad fixing unit 410, a reaction pad fixing unit 420, a detection pad 430, and an absorption pad 440, as shown in FIG. ) is provided, the sample input pad 411 is fixed to the sample input pad fixing part 410, the reaction pad 421 is fixed to the reaction pad fixing part 420, and then the sample is placed on the sample input pad 411. It may be a lateral flow sensor 400 using a strip formed so that when the liquid is injected, the sample liquid passes through the reaction pad 421 and passes the test line 431 and the control line 432.
- the upper strip of FIG. 10 shows a reaction pad containing the secondary aptamer of the present invention to which the marker is bound, a detection line to which the primary aptamer specifically binding to the target substance is immobilized, and DNA binding to the secondary aptamer.
- a lateral flow sensor with a fixed control line is shown.
- the target substance detection method according to the present invention is a detection method using the aptamer according to the present invention, wherein the primary aptamer-target formed by the binding of the target substance and the primary aptamer that binds specifically to the target substance It is characterized by using a secondary aptamer that specifically binds to the material complex.
- the target material detection method may include contacting a sample suspected of having a target material with the primary aptamer and the secondary aptamer simultaneously or with a time difference.
- the sample may be contacted with a primary aptamer and then contacted with a secondary aptamer, contacted with a secondary aptamer and then contacted with a primary aptamer, or simultaneously contacted with a primary aptamer and a secondary aptamer. Regardless of the order, if the target substance is present in the sample, when the target substance meets the primary aptamer, a primary aptamer-target substance complex is formed, whereas if the target substance does not exist in the sample, the primary aptamer-target substance is formed. No material complexes are formed.
- the target substance when the target substance is present in the sample regardless of the order, when the first aptamer-target substance complex is exposed to the second aptamer, a first aptamer-target substance-second aptamer complex is formed, whereas in the sample When the target substance does not exist, the primary aptamer-target substance-secondary aptamer complex is not formed.
- At least one of the primary aptamer and the secondary aptamer may be an immobilized aptamer previously immobilized on a support.
- the immobilized aptamer may be referred to as an immobilized aptamer or a capture aptamer
- the non-immobilized aptamer may be referred to as a non-immobilized aptamer or a detection aptamer.
- the primary aptamer and the secondary aptamer may be non-anchored aptamers that are not previously immobilized on a support. In this case, contacting a sample suspected of having a target substance with the non-immobilized primary aptamer and the non-immobilized secondary aptamer at the same time or with a time difference; and immobilizing at least one of the primary aptamer and the secondary aptamer to the support.
- a non-fixed primary aptamer, a secondary aptamer, or a mixture thereof is prepared at the front of the diagnostic strip, and the sample is After injecting and contacting them, it may be fixed in such a way as to induce binding between the support and the primary aptamer or the secondary aptamer when reaching a specific region such as a detection region by flow.
- at least one of the primary aptamer and the secondary aptamer may be modified to have a functional group capable of binding to the support.
- the support may be modified to bind to at least one of the primary aptamer and the secondary aptamer.
- the bond between the aptamer and the support may be a covalent bond, a non-covalent bond, or an electrostatic bond.
- the first aptamer-target substance complex and the second aptamer complex are formed.
- a step of determining whether a primary aptamer-target substance-secondary aptamer complex formed by binding of secondary aptamers is formed and, accordingly, determining the presence or absence of the target substance in the sample may be performed.
- the primary aptamer-target material-secondary aptamer complex means that a complex is formed due to the bond formed between the primary aptamer, the target material, and the secondary aptamer, and the binding structure between them is defined. It is not.
- Whether or not the first aptamer-target substance-secondary aptamer complex is formed can be determined through PCR. Specifically, first, after conjugating a target material to a magnetic bead, a primary aptamer is reacted. Next, after removing the unreacted primary aptamer by centrifugation, the secondary aptamer is added and reacted. Thereafter, unreacted secondary aptamers can be removed by centrifugation, and then PCR can be performed using primers recognizing the secondary aptamers bound to the beads. At this time, beads without a target material may be used as a comparison group.
- the primary aptamer or secondary aptamer may be labeled with a signal generating substance.
- the primary aptamer or secondary aptamer may be labeled with a material selected from the group consisting of quantum dots, fluorescent substances, luminescent substances, staining reagents, enzyme dyes (HRP), and mixtures thereof.
- labeling the unimmobilized mobile aptamer with a signal generating material is associated with signal generation in the detection zone depending on the presence or absence of the target material. This is preferable because differences may occur.
- Test Example 1 Confirmation of non-specific binding of primary aptamers and secondary aptamers
- the secondary aptamer 200 labeled with biotin at the end was applied to the plate 300 on which is immobilized.
- streptavidin-HRP Haseradish Peroxidase
- Test Example 2 Confirmation of binding affinity of the secondary aptamer to the primary aptamer-target material complex
- Example 2 In order to confirm the binding affinity of the secondary aptamer selected in Example 1 to the primary aptamer-target material complex, the primary aptamer and the target-bound form and the target-only form of the secondary aptamer The degree of binding was confirmed.
- the primary aptamer (DEHP-57 Aptamer) (primary aptamer concentration: 200 pmole/100 ⁇ l, 2 ⁇ M) is brought into contact with the target material, DEHP, and DEHP beads composed of beads to form a primary aptamer. Formation of the aptamer-target material complex was induced, and after the introduction of DEHP 2nd Aptamer, the fluorescence value was measured through HRP labeled at the end of the DEHP 2nd aptamer.
- DEHP-57 Aptamer primary aptamer concentration: 200 pmole/100 ⁇ l, 2 ⁇ M
- the primary aptamer does not exist (primary aptamer concentration of 0 pmole)
- the DEHP bead and the DEHP secondary aptamer are brought into contact to measure fluorescence through HRP labeled at the end of the DEHP secondary aptamer. measured.
- Types of secondary aptamers Fluorescence measurement (RLU: relative luminescence units) Primary aptamer concentration: 200 pmoles Primary aptamer concentration: 0 pmole S008-A1-1 202700 34590 S008-A1-2 34210000 23250 S008-A1-3 34380 51440 S008-A1-4 264200 24230 S008-A1-5 28290000 40970 S008-A1-6 13050000 41750 S008-A1-7 60700 24350 S008-A1-8 174200 60460 S008-A1-9 202000000 179900 Library (negative control) 219100 56200
- the fluorescence measurement value was significantly higher than in the case where it was not, and in all cases where it was not, it was judged as negative. It was confirmed that the complex was recognized and bound only in the state.
- the secondary aptamer according to the present invention has very low binding ability to the primary aptamer or target substance, and significantly higher binding to the primary aptamer-target substance complex, so it can be used very effectively for detecting the target substance. will be.
- Test Example 3 Sandwich assay for target substance DEHP
- the primary aptamer (T17) is immobilized on the surface by BSA on the surface and covalently bonded to BSA using the Thiol group of the primary aptamer, and then reacted with the primary aptamer by applying DEHP, a target material, and labeled with biotin.
- Secondary aptamers B7, B30, B31, B32, and B33 were applied, respectively (FIG. 6).
- the degree of recognition and binding of the secondary aptamer to the primary aptamer-target material complex was measured using streptavidin-HRP, and the results are shown in FIG. 7 .
- the first aptamer, T17 was immobilized on the surface and the sandwich assay was performed with the second aptamer, B33, the highest signal/noise value (S/N ratio) was 3.5.
- Example 2 Selection of aptamers that bind to the primary aptamer and the target complex - 2 nd aptamer SELEX
- a primary aptamer is immobilized on a surface (plate, bead, etc.), and a binding reaction is induced to a random library having a random base sequence in a state where a target is formed as a primary aptamer complex. Thereafter, those that do not bind are removed by washing, and finally, the aptamer that binds to the primary aptamer and the target complex is finally selected.
- the Biotin-labeled primary aptamer selected by the general SELEX method is immobilized on a streptavidin-coated plate. After that, the unbound ones are removed by washing, and the COVID19 N protein at a concentration of 0.5 uM is combined with the primary aptamer to form the primary aptamer target complex. Then, in order to select the secondary aptamer, the library having the random base sequence N40 was treated with the target-free primary aptamer immobilized plate well (plate No. 1) to remove the library that reacts with the primary aptamer, and the N protein The library that reacts to N protein is removed by reacting sup of plate No. 1 on the fixed plate using His tag.
- the library obtained here is finally reacted with the primary aptamer target complex to select secondary aptamers that bind thereto (FIG. 8).
- the primary aptamer target complex to select secondary aptamers that bind thereto (FIG. 8).
- 2nd aptamers (2nd Aptamer 1-4) having different base sequences were selected.
- Test Example 4 Verification of secondary aptamer binding force
- Example 2 2 libraries and 4 2 nd aptamers selected in Example 2 were mixed at a concentration of 10 pmoles/well in a buffer solution (40 mM HEPES pH 7.5, 102 mM NaCl, 5 mM KCl, 5 mM MgCl 2 and 0.05% Tween- 20), heated at 95 °C, and slowly lowered the temperature to room temperature to stabilize the structure.
- a buffer solution 40 mM HEPES pH 7.5, 102 mM NaCl, 5 mM KCl, 5 mM MgCl 2 and 0.05% Tween- 20
- the biotin-coupled aptamer was mixed with the target protein to proceed with the binding reaction, and 10 ul of Dynal His-tag bead was added to the binding reaction at RT for 30 minutes and the target was immobilized. After that, it was washed three times with 100 ul of the buffer solution.
- Bio-rad Substrates A and B were mixed and added to wells by 100 ul, and luminescence was measured with an ELISA reader (Table 3 and FIG. 9), and the binding strength of all four aptamers was measured to be around 1 nM.
- Test Example 5 Preparation of lateral flow sensor and comparative evaluation for selection of primary aptamer secondary aptamer pair
- a sample input pad fixing part 410 As shown in FIG. 10, a sample input pad fixing part 410, a reaction pad fixing part 420, a detection pad 430, and an absorption pad 440 are provided, and the sample input pad fixing part 410 has a sample input pad ( 411) is fixed, the reaction pad 421 is fixed to the reaction pad fixing part 420, and then the sample solution is injected into the sample input pad 411, and the sample solution passes through the reaction pad 421 to the detection line (Test).
- a lateral flow sensor 400 which is a diagnostic kit, was manufactured by using a strip formed to pass the line 431 and the control line 432.
- the aptamer In order to conjugate the aptamer to the quantum dot and the quantum dot bead, it was synthesized by conjugating a thiol group to the 5 prime of the aptamer. Quantum dots were mixed with thiol-aptamer, reacted for 16 hours, put into a MWCO 100 kDa tube, centrifuged at 6,000 rpm for 10 minutes, and washed three times using DI water. In the case of quantum dot beads, Bovine serum albumin (BSA ) were combined by an electrostatic method, and thiol-aptamer was mixed to form an S-S bond with the thiol group in the amino acid of BSA to proceed with conjugation.
- BSA Bovine serum albumin
- cytosine (C) 10mer was extended to 3' of the primary aptamer, and Biotin-guanine (G) 10mer was fixed to the membrane control line using SA.
- the strength of the test line according to the addition of the target protein (N protein) was compared to compare the aptamer pair wanted to select.
- N protein target protein
- Test Example 6 Comparison of sensitivity evaluation using gold nanoparticles and quantum dots
- Test Example 5 after manufacturing a lateral flow sensor using gold nanoparticles instead of quantum dots or quantum dot beads, the sensitivity of the diagnostic kit using gold nanoparticles and quantum dots was compared and evaluated.
- kits using gold nanoparticles as a result of testing for each N-protein concentration, up to 10 ug/mL concentration was visually confirmed.
- kits using quantum dots and quantum dot beads were able to visually confirm up to 5 ug/mL concentration, and as a result of measurement using a fluorescence reader, detection was possible up to 1 ug/mL concentration (FIGS. 12 and 13).
Abstract
The present invention relates to a secondary aptamer that binds specifically to a primary aptamer-target material complex that is formed as a target material and a primary aptamer binding specifically to the target material are coupled to each other. In addition, the present invention relates to a method for detecting a target material, using the secondary aptamer and a diagnostic kit comprising the secondary aptamer.
Description
본 발명은, 압타머 및 이를 포함하는 진단키트에 관한 것으로, 특히, 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머에 관한 것이다. The present invention relates to an aptamer and a diagnostic kit including the same, and in particular, a target substance and a primary aptamer that specifically binds to the target substance are formed by binding to a primary aptamer-target substance complex and specific binding It is about a secondary aptamer that does.
전염병은 국가를 불문하고 세계적으로 유행하여 인류의 건강을 위협하고 있으며 그 위험성이 더욱 심화되고 있다. 사스, 신종 인플루엔자에 이어 최근 COVID-19 바이러스의 대유행이 이어지고 있으며 바이러스 감염여부를 검사하기 위한 다양한 방법들이 제시되었다. 검사방법은 크게 생체 시료 채취 후 핵산 증폭을 통해 바이러스 감염여부를 확인하는 PCR 검사, 항체가 형성되었는지 검사하는 항체검사, 바이러스를 직접 검출하는 항원검사로 분류되어 각 분야에서 활발한 연구가 이어지고 있다.Infectious diseases are prevalent all over the world regardless of country, threatening human health, and the risk is intensifying. Following SARS and swine flu, the recent COVID-19 virus pandemic continues, and various methods have been proposed to test for viral infection. Test methods are largely classified into PCR tests to check for virus infection through nucleic acid amplification after collecting biological samples, antibody tests to check whether antibodies are formed, and antigen tests to directly detect viruses, and active research is continuing in each field.
압타머는 DNA 또는 RNA로 이루어진 핵산 물질로서 단백질, 세포, 미생물 등 특정물질을 표적으로 하여 특이적으로 결합할 수 있다. 압타머는 짧은 길이의 핵산 서열로 항체에 비해 안정성이 우수하여 장기간 보존이 가능하고 표적에 대해 높은 친화성을 가지는 장점이 있어 다양한 기술분야에서 응용 가능한 각광받는 물질이다. 이러한 압타머의 우수성에 의해 압타머를 감염병 검사법에 응용하려는 움직임이 있으며, 바이러스의 두 부위에 결합하는 압타머 쌍을 바이러스에 결합시키고 형광물질 등 이를 표지하는 표지체를 통해 바이러스 검출 여부를 검출하는 방법이 알려져 있다.Aptamers are nucleic acid materials composed of DNA or RNA, and can specifically bind to specific substances such as proteins, cells, and microorganisms. Aptamers are short-length nucleic acid sequences that are superior in stability to antibodies, enabling long-term preservation and having high affinity for a target, and thus are in the spotlight for application in various technical fields. Due to the excellence of these aptamers, there is a movement to apply aptamers to infectious disease testing methods, and a pair of aptamers that bind to two parts of the virus is bound to the virus and detects whether the virus is detected through a marker that labels it, such as a fluorescent substance. method is known.
이와 관련하여 중국공개특허 106443003 호는 형광 수용체로 표지된 압타머를 접합패드에 부착하고, 형광 물질로 표지된 프로브를 검출라인에 부착하여 타겟 물질의 존재 여부에 따라 이들 사이에 FRET 현상이 일어나도록 한 형광 소광 테스트 스트립에 대하여 제시하고 있다. 또한 한국공개특허 2017-0103119 호는 형광 염색약이 인터칼레이팅되어 있는 DNA 압타머 복합체를 지지체에 고정되어 있는 상보적인 가닥에 이중나선을 형성하도록 결합시켜 놓고, 표적물질이 존재할 경우 DNA 압타머 복합체가 표적물질과 결합하여 떨어져 나감에 따라 방출된 형광 염색화를 시각화하여 검출하는 방법에 대하여 제시하고 있다. In this regard, Chinese Patent Publication No. 106443003 discloses that an aptamer labeled with a fluorescent receptor is attached to a conjugation pad, and a probe labeled with a fluorescent material is attached to a detection line so that a FRET phenomenon occurs between them depending on the presence or absence of a target material. A fluorescence quenching test strip is presented. In addition, Korean Patent Publication No. 2017-0103119 discloses that a DNA aptamer complex intercalated with a fluorescent dye is bound to a complementary strand fixed to a support to form a double helix, and when a target substance is present, the DNA aptamer complex A method for visualizing and detecting fluorescence staining emitted as it binds to and detaches from a target material is proposed.
그러나 여전히 압타머 등을 이용한 진단키트의 민감도 및 특이도가 충분치 않은 실정이다.However, the sensitivity and specificity of diagnostic kits using aptamers and the like are still insufficient.
본 발명의 압타머는 상기와 같은 문제점을 해결하기 위하여 안출된 것으로서, 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머인, 압타머를 제공하는 것을 그 목적으로 한다.The aptamer of the present invention was devised to solve the above problems, and specifically binds to the target substance and the primary aptamer-target substance complex formed by binding the target substance and the primary aptamer that binds specifically to the target substance. Its purpose is to provide an aptamer, which is a secondary aptamer.
또한, 본 발명은 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머; 및 상기 2차 압타머에 결합되어 표지하는 표지체;를 포함하는 압타머 복합체를 제공하는 것을 또 다른 목적으로 한다.In addition, the present invention is a primary aptamer formed by combining a target substance and a primary aptamer that specifically binds to the target substance - a secondary aptamer that specifically binds to the target substance complex; Another object is to provide an aptamer complex comprising a; and a label that binds to and labels the secondary aptamer.
또한, 본 발명은 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머를 포함하는 진단키트를 제공하는 것을 또 다른 목적으로 한다.In addition, the present invention provides a diagnostic kit including a target substance and a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding of a primary aptamer that specifically binds to the target substance. that serves another purpose.
또한, 본 발명은 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머를 이용하는 표적물질 검출방법을 제공하는 것을 또 다른 목적으로 한다.In addition, the present invention provides a target substance detection method using a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding a target substance and a primary aptamer that specifically binds to the target substance to do for another purpose.
본 발명은 또한 상기한 명확한 목적 이외에 이러한 목적 및 본 명세서의 전반적인 기술로부터 이 분야의 통상인에 의해 용이하게 도출될 수 있는 다른 목적을 달성함을 그 목적으로 할 수 있다.The present invention may also have as its object to achieve other objects that can be easily derived by a person of ordinary skill in the art from these objects and the overall description of this specification in addition to the above clear objects.
본 발명의 압타머는 상술한 바와 같은 목적을 달성하기 위하여, 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머인 것을 특징으로 한다.In order to achieve the above object, the aptamer of the present invention is formed by combining a target substance and a primary aptamer that specifically binds to the target substance, and a secondary aptamer-target substance complex that specifically binds to the target substance. It is characterized in that it is an aptamer.
또한, 상기 표적물질은 항원, 항체, 바이러스, 올리고뉴클레오티드, 단백질, 탄수화물, 과당, 글리코당, 올리고당, 호르몬, 수용체, 펩타이드, 기질, 대사물질, 전이상태 유사물, 보조인자, 저해제, 약물, 염료, 영양분, 성장인자, 조직, 무기 유기 영양소, 유해 환경물질, 또는 당단백질의 올리고당일 수 있다.In addition, the target substance is an antigen, antibody, virus, oligonucleotide, protein, carbohydrate, fructose, glycosaccharide, oligosaccharide, hormone, receptor, peptide, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye , nutrients, growth factors, tissues, inorganic organic nutrients, harmful environmental substances, or oligosaccharides of glycoproteins.
그리고, 상기 2차 압타머는 상기 1차 압타머와 상기 표적물질이 결합함으로써 형성된 3차원 구조를 인식할 수 있다.In addition, the secondary aptamer can recognize a three-dimensional structure formed by binding the primary aptamer and the target material.
그리고, 상기 2차 압타머는 상기 표적물질과 특이적 결합을 할 수 있다.And, the secondary aptamer can specifically bind to the target material.
그리고, 상기 2차 압타머는 상기 1차 압타머-표적물질 복합체에 대한 결합 친화성이 상기 표적물질에 대한 결합 친화성에 대해 2 내지 2000 배, 2 내지 1800 배, 또는 2 내지 1500 배일 수 있다.In addition, the binding affinity of the secondary aptamer to the primary aptamer-target material complex may be 2 to 2000 times, 2 to 1800 times, or 2 to 1500 times the binding affinity to the target material.
한편, 본 발명에 따른 압타머 복합체는, 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머; 및 상기 2차 압타머에 결합되어 표지하는 표지체;를 포함하는 것을 특징으로 한다.Meanwhile, the aptamer complex according to the present invention includes a primary aptamer formed by binding a target substance and a primary aptamer specifically binding to the target substance, and a secondary aptamer specifically binding to the target substance complex; and a label that binds to and labels the secondary aptamer.
또한, 상기 표지체는 형광체, 발광체, 양자점, 양자점 비드, 골드 나노 파티클, 유로피움, 염료, 방사성 표지, 전기화학적 작용기, 효소, 효소기질, 및 이들의 혼합물로 이루어진 군으로부터 선택될 수 있다.In addition, the marker may be selected from the group consisting of a fluorescent substance, a luminous substance, a quantum dot, a quantum dot bead, gold nanoparticles, europium, a dye, a radioactive label, an electrochemical functional group, an enzyme, an enzyme substrate, and mixtures thereof.
그리고, 상기 표지체는 카드뮴설파이드(CdS), 카드뮴셀레나이드(CdSe), 카드뮴텔레나이드(CdTe), 징크설파이드(ZnS), 징크셀레나이드(ZnSe), 징크텔레나이드(ZnTe), 징크옥사이드(ZnO), 징크옥사이드징크셀레나이드(ZnOZnSe), 징크옥사이드징크설파이드(ZnOZnS), 카드뮴옥사이드(CdO), 카드뮴셀레늄설파이드(CdSeS), 카드뮴셀레늄텔레나이드(CdSeTe), 카드뮴설파이드텔레나이드(CdSTe), 카드뮴징크설파이드(CdZnS), 카드뮴징크셀레나이드(CdZnSe), 카드뮴징크텔레나이드(CdZnTe), 징크셀레늄설파이드(ZnSeS), 징크셀레늄텔레나이드(ZnSeTe), 징크설파이드텔레나이드(ZnSTe), 카드뮴징크옥사이드(CdZnO), 징크셀레늄옥사이드(ZnSeO), 카드뮴셀레늄(CdSe), 카드뮴징크셀레늄설파이드(CdZnSeS), 카드뮴징크셀레늄텔레나이드(CdZnSeTe), 카드뮴셀레나이드징크설퍼(CdSeZnS), 카드뮴징크설파이드텔레나이드(CdZnSTe), 갈륨포스포러스(GaP), 갈륨아세나이드(GaAs), 갈륨니트라이드(GaN), 인듐포스포러스니트라이드(InPN), 인듐아세나이드니트라이드(InAsN), 인듐갈륨포스포러스(InGaP), 인듐갈륨아세나이드(InGaAs), 인듐갈륨니트라이드(InGaN), 인듐아세나이드니트라이드(InAsN), 인듐갈륨포스포러스(InGaP), 인듐알루미늄포스포러스안티모니(InAlPSb), 인듐갈륨포스포러스징크설파이드(InGaPZnS), 인듐갈륨포스포러스징크셀레나이드(InGaPZnSe), 인듐포스포러스징크셀레나이드(InPZnSe), 인듐포스포러스징크설파이드(InPZnS), 및 이들의 혼합물로 이루어진 군으로부터 선택될 수 있다.In addition, the marker is cadmium sulfide (CdS), cadmium selenide (CdSe), cadmium tellenide (CdTe), zinc sulfide (ZnS), zinc selenide (ZnSe), zinc tellenide (ZnTe), zinc oxide (ZnO ), zinc oxide zinc selenide (ZnOZnSe), zinc oxide zinc sulfide (ZnOZnS), cadmium oxide (CdO), cadmium selenium sulfide (CdSeS), cadmium selenium tellenide (CdSeTe), cadmium sulfide tellenide (CdSTe), cadmium zinc Sulfide (CdZnS), Cadmium Zinc Selenide (CdZnSe), Cadmium Zinc Tellenide (CdZnTe), Zinc Selenium Sulfide (ZnSeS), Zinc Selenium Tellenide (ZnSeTe), Zinc Sulfide Tellenide (ZnSTe), Cadmium Zinc Oxide (CdZnO) , zinc selenium oxide (ZnSeO), cadmium selenium (CdSe), cadmium zinc selenium sulfide (CdZnSeS), cadmium zinc selenium tellenide (CdZnSeTe), cadmium selenide zinc sulfur (CdSeZnS), cadmium zinc selenium tellenide (CdZnSTe), gallium Phosphorus (GaP), Gallium Arsenide (GaAs), Gallium Nitride (GaN), Indium Phosphorus Nitride (InPN), Indium Arsenide Nitride (InAsN), Indium Gallium Phosphorus (InGaP), Indium Gallium Arsenide (InGaAs), indium gallium nitride (InGaN), indium arsenide nitride (InAsN), indium gallium phosphorus (InGaP), indium aluminum phosphorus antimony (InAlPSb), indium gallium phosphorus zinc sulfide (InGaPZnS), indium It may be selected from the group consisting of gallium phosphorus zinc selenide (InGaPZnSe), indium phosphorus zinc selenide (InPZnSe), indium phosphorus zinc sulfide (InPZnS), and mixtures thereof.
그리고, 상기 표지체는 상기 2차 압타머와 공유결합, 이온결합, 또는 정전기적 결합을 통해 결합을 형성할 수 있다.In addition, the marker may form a bond with the secondary aptamer through a covalent bond, an ionic bond, or an electrostatic bond.
그리고, 상기 표지체는 상기 2차 압타머와 EDC 또는 EDC/NHS(또는 sulfo-NHS) 반응을 이용한 결합, 디설파이드 결합(S-S), 또는 말레이미드(maleimide)를 이용한 결합을 통해 결합을 형성할 수 있다.In addition, the marker may form a bond with the secondary aptamer through a bond using an EDC or EDC / NHS (or sulfo-NHS) reaction, a disulfide bond (S-S), or a bond using maleimide. have.
한편, 본 발명에 따른 진단키트는, 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머를 포함하는 것을 특징으로 한다.On the other hand, the diagnostic kit according to the present invention includes a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding a target substance and a primary aptamer that specifically binds to the target substance. characterized by
그리고, 상기 진단키트는 측면 유동 진단 스트립(LFA), 효소면역검출키트(ELISA), 또는 바이오칩일 수 있다.In addition, the diagnostic kit may be a lateral flow diagnostic strip (LFA), an enzyme immunoassay kit (ELISA), or a biochip.
또한, 상기 진단키트는 표적물질과 특이적 결합을 하는 1차 압타머를 포함할 수 있다.In addition, the diagnostic kit may include a primary aptamer that specifically binds to a target substance.
또한, 상기 1차 압타머 및 2차 압타머 중 적어도 하나는 지지체에 고정된 것일 수 있다.In addition, at least one of the primary aptamer and the secondary aptamer may be immobilized to the support.
또한, 상기 1차 압타머 및 2차 압타머는 비고정식일 수 있다.In addition, the primary aptamer and the secondary aptamer may be non-fixed.
한편, 본 발명에 따른 표적물질 검출방법은, 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머를 이용하는 것을 특징으로 한다.On the other hand, the method for detecting a target substance according to the present invention uses a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding a target substance and a primary aptamer that specifically binds to the target substance. characterized by use.
그리고, 상기 1차 압타머 또는 2차 압타머는 신호발생물질로 표지된 것일 수 있다.In addition, the primary aptamer or the secondary aptamer may be labeled with a signal generating substance.
그리고, 상기 1차 압타머 또는 2차 압타머는 형광체, 발광체, 염색시약, 효소 염색(HRP) 및 이들의 혼합물로 이루어진 군으로부터 선택된 것으로 표지된 것일 수 있다.In addition, the primary aptamer or secondary aptamer may be labeled with a substance selected from the group consisting of a fluorescent substance, a luminescent substance, a staining reagent, an enzyme dye (HRP), and a mixture thereof.
또한, 상기 표적물질 검출방법은 표적물질이 존재할 것으로 의심되는 시료를 상기 1차 압타머 및 상기 2차 압타머와 동시에 또는 시간차를 두고 접촉시키는 단계를 포함할 수 있다. In addition, the target material detection method may include contacting a sample suspected of having a target material with the primary aptamer and the secondary aptamer simultaneously or with a time difference.
또한, 상기 1차 압타머 및 2차 압타머 중 적어도 하나는 지지체에 고정된 것일 수 있다.In addition, at least one of the primary aptamer and the secondary aptamer may be immobilized to the support.
또한, 상기 표적물질 검출방법은, 표적물질이 존재할 것으로 의심되는 시료를 비고정 1차 압타머 및 비고정 2차 압타머와 동시에 또는 시간차를 두고 접촉시키는 단계; 및 상기 1차 압타머 및 상기 2차 압타머 중 적어도 하나가 지지체에 고정되는 단계;를 포함할 수 있다.In addition, the target material detection method may include contacting a sample suspected of having a target material with a non-fixed primary aptamer and a non-fixed secondary aptamer at the same time or with a time difference; and immobilizing at least one of the primary aptamer and the secondary aptamer to the support.
그리고, 상기 1차 압타머 및 2차 압타머 중 적어도 하나가 지지체와 결합가능한 작용기를 가질 수 있다.In addition, at least one of the primary aptamer and the secondary aptamer may have a functional group capable of binding to the support.
또한, 상기 표적물질 검출방법은, 상기 1차 압타머-표적물질 복합체와 상기 2차 압타머가 결합하여 형성된 1차 압타머-표적물질-2차 압타머 복합체의 형성 여부에 따라 시료 내 표적물질의 존부를 판단하는 단계를 포함할 수 있다.In addition, the target substance detection method determines whether the target substance in the sample is formed according to whether a first aptamer-target substance-second aptamer complex formed by binding the first aptamer-target substance complex and the second aptamer is formed. It may include a step of determining existence or nonexistence.
또한, 상기 1차 압타머-표적물질-2차 압타머 복합체의 형성 여부는 PCR을 통해 판단할 수 있다.In addition, whether or not the first aptamer-target substance-secondary aptamer complex is formed can be determined through PCR.
또한, 상기 1차 압타머-표적물질-2차 압타머 복합체의 형성 여부는 시각적 신호를 통해 판단할 수 있다.In addition, whether or not the first aptamer-target substance-secondary aptamer complex is formed can be determined through a visual signal.
본 발명에 따른 압타머 및 이를 이용한 표적물질 검출방법은 검출 감도가 우수하고 조작이 간편하며, 표적물질에 결합가능한 2차 시약에 대하여 표지화가 불필요하다. 또한 항체를 대체하여 압타머를 사용함으로써 소분자 검출이 용이하고, 특이도를 상승시킬 수 있다.The aptamer and the target substance detection method using the same according to the present invention have excellent detection sensitivity, simple operation, and do not require labeling of a secondary reagent capable of binding to the target substance. In addition, by using an aptamer instead of an antibody, small molecule detection can be facilitated and specificity can be increased.
도 1은 본 발명의 일 실시예에 따른 압타머를 도식화한 것이다. 1 is a schematic diagram of an aptamer according to an embodiment of the present invention.
도 2는 본 발명의 일 실시예에 따른 1차 압타머와 2차 압타머의 비특이 결합 확인을 위한 어쎄이의 모식도이다.2 is a schematic diagram of an assay for confirming non-specific binding between a primary aptamer and a secondary aptamer according to an embodiment of the present invention.
도 3은 본 발명의 일 실시예에 따른 1차 압타머와 2차 압타머의 비특이 결합 확인을 위한 테스트 결과이다.3 is a test result for confirming non-specific binding between a primary aptamer and a secondary aptamer according to an embodiment of the present invention.
도 4는 본 발명의 일 실시예에 따른 2차 압타머의 1차 압타머-표적물질 복합체에 대한 결합 과정 모식도이다.4 is a schematic diagram of a process for binding a secondary aptamer to a primary aptamer-target material complex according to an embodiment of the present invention.
도 5는 본 발명의 일 실시예에 따른 2차 압타머의 1차 압타머-표적물질 복합체에 대한 결합 친화성 테스트 결과이다.5 is a binding affinity test result of a secondary aptamer to a primary aptamer-target material complex according to an embodiment of the present invention.
도 6은 본 발명의 일 실시예에 따른 1차 압타머와 2차 압타머의 샌드위치 방식의 어쎄이의 모식도이다. 6 is a schematic diagram of a sandwich method assay of a primary aptamer and a secondary aptamer according to an embodiment of the present invention.
도 7은 본 발명의 일 실시예에 따른 1차 압타머와 2차 압타머의 샌드위치 방식의 어쎄이의 결과이다.7 is a result of a sandwich method assay of a primary aptamer and a secondary aptamer according to an embodiment of the present invention.
도 8은 본 발명의 일 실시예에 따른 2차 압타머의 선별 방법에 대한 모식도이다.8 is a schematic diagram of a method for screening secondary aptamers according to an embodiment of the present invention.
도 9는 본 발명의 일 실시예에 따른 2차 압타머의 COVID19 N protein에 대한 결합력을 측정한 결과이다.9 is a result of measuring the binding force of the secondary aptamer to the COVID19 N protein according to an embodiment of the present invention.
도 10은 본 발명의 일 실시예에 따른 진단키트인 측방유동센서의 구조 및 작용를 나타낸 것이다.10 shows the structure and operation of a lateral flow sensor, which is a diagnostic kit according to an embodiment of the present invention.
도 11은 본 발명의 일 실시예에 따른 1차 압타머-2차 압타머 페어를 선정하기 위한 측방유동센서 테스트 결과이다.11 is a lateral flow sensor test result for selecting a primary aptamer-secondary aptamer pair according to an embodiment of the present invention.
도 12는 본 발명의 일 실시예에 따른 진단키트 3 종의 민감도 테스트 결과이다.12 is a sensitivity test result of three diagnostic kits according to an embodiment of the present invention.
도 13은 본 발명의 일 실시예에 따른 진단키트의 타겟 농도에 따른 형광리더기 측정 결과이다.13 is a fluorescence reader measurement result according to a target concentration of a diagnostic kit according to an embodiment of the present invention.
이하, 본 발명의 바람직한 실시예에 대하여 상세히 설명한다. Hereinafter, preferred embodiments of the present invention will be described in detail.
다만, 아래는 특정 실시예들을 예시하여 상세히 설명하는 것일 뿐, 본 발명은 다양하게 변경될 수 있고 여러 가지 형태를 가질 수 있기 때문에, 예시된 특정 실시예들에 본 발명이 한정되는 것은 아니다. 본 발명은 본 발명의 사상 및 기술 범위에 포함되는 모든 변경, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다.However, the following is only a detailed description by exemplifying specific embodiments, and since the present invention may be variously changed and may have various forms, the present invention is not limited to the specific embodiments illustrated. It should be understood that the present invention includes all modifications, equivalents and substitutes included in the spirit and scope of the present invention.
또한, 하기의 설명에서는 구체적인 구성요소 등과 같은 많은 특정사항들이 설명되어 있는데, 이는 본 발명의 보다 전반적인 이해를 돕기 위해서 제공된 것일 뿐 이러한 특정 사항들 없이도 본 발명이 실시될 수 있음은 이 기술분야에서 통상의 지식을 가진 자에게는 자명하다 할 것이다. 그리고, 본 발명을 설명함에 있어서, 관련된 공지 기능 혹은 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.In addition, in the following description, many specific details such as specific components are described, which are provided to help a more general understanding of the present invention, and it is common in the art that the present invention can be practiced without these specific details. It will be self-evident to those who have the knowledge of And, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted.
그리고, 본 출원에서 사용한 용어는 단지 특정한 실시예를 설명하기 위해 사용된 것으로, 본 발명을 한정하려는 의도가 아니다. 다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥 상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.And, the terms used in this application are only used to describe specific embodiments, and are not intended to limit the present invention. Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't
본 출원에서, 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다.In this application, expressions in the singular number include plural expressions unless the context clearly dictates otherwise.
본 출원에서, 제1, 제2 등의 용어는 다양한 구성요소들을 설명하는데 사용될 수 있지만, 상기 구성요소들은 상기 용어들에 의해 한정되어서는 안 된다. 상기 용어들은 하나의 구성요소를 다른 구성요소로부터 구별하는 목적으로만 사용된다. 예를 들어, 본 발명의 권리 범위를 벗어나지 않으면서 제1 구성요소는 제2 구성요소로 명명될 수 있고, 유사하게 제2 구성요소도 제1 구성요소로 명명될 수 있다.In this application, terms such as first and second may be used to describe various components, but the components should not be limited by the terms. These terms are only used for the purpose of distinguishing one component from another. For example, a first element may be termed a second element, and similarly, a second element may be termed a first element, without departing from the scope of the present invention.
본 출원에서, '포함하다', '함유하다' 또는 '가지다' 등의 용어는 명세서 상에 기재된 특징, 구성요소(또는 구성성분) 등이 존재함을 지칭하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 구성요소 등이 존재하지 않거나 부가될 수 없음을 의미하는 것은 아니다.In this application, terms such as 'comprise', 'include' or 'having' are intended to indicate that the features, components (or components), etc. described in the specification exist, but one or more other features or It does not mean that components or the like do not exist or cannot be added.
압타머(Aptamer)는 표적물질에 특이적으로 결합하는 단일가닥핵산(DNA, RNA, 또는 변형핵산)으로서, 항체에 비해 안정성이 높아 진단 시스템에서의 활용성이 높고 크기가 작아 변형이 용이하여 바이러스 등의 변이체에 대하여 대응이 용이하다. Aptamer is a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that specifically binds to a target substance. It is easy to respond to variants such as
본 발명의 압타머는, 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머인 것을 특징으로 한다. The aptamer of the present invention is characterized in that it is a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding a target substance and a primary aptamer that specifically binds to the target substance.
상기 표적물질은 항원, 항체, 바이러스, 올리고뉴클레오티드, 단백질, 탄수화물, 과당, 글리코당, 올리고당, 호르몬, 수용체, 펩타이드, 기질, 대사물질, 전이상태 유사물, 보조인자, 저해제, 약물, 염료, 영양분, 성장인자, 조직, 무기 유기 영양소, 유해 환경물질, 또는 당단백질의 올리고당일 수 있다.The target substance is an antigen, antibody, virus, oligonucleotide, protein, carbohydrate, fructose, glycosaccharide, oligosaccharide, hormone, receptor, peptide, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient , growth factors, tissues, inorganic organic nutrients, harmful environmental substances, or oligosaccharides of glycoproteins.
상기 1차 압타머는 표적물질과 특이적 결합을 하는 압타머이다.The primary aptamer is an aptamer that specifically binds to a target substance.
상기 2차 압타머는 단독의 표적물질과 특이적 결합을 하지 않는 것일 수 있다. 또한, 상기 2차 압타머 또한 1차 압타머와 같이 상기 표적물질과 결합 친화성이 있거나 특이적 결합을 할 수 있으나, 이 경우 2차 압타머는 1차 압타머-표적물질 복합체에 대한 결합 친화성이 표적물질에 대한 결합 친화성보다 현저히 높은 것을 특징으로 할 수 있다. 이는 2차 압타머의 타겟이 단순 표적물질이 아닌 1차 압타머-표적물질 복합체임을 의미할 수 있다. 표적물질의 검출에 표적물질의 서로 다른 두 결합 부위에 결합하는 압타머쌍을 이용하는 것에 비해 본 발명에 따르면 하나의 압타머가 다른 압타머와 표적물질의 결합체를 인식하므로 특이도와 민감도를 매우 향상시킬 수 있다.The secondary aptamer may not specifically bind to a single target substance. In addition, the secondary aptamer also has binding affinity to the target substance or can bind specifically to the target substance like the primary aptamer, but in this case, the secondary aptamer has binding affinity for the primary aptamer-target substance complex It can be characterized as significantly higher than the binding affinity for the target substance. This may mean that the target of the secondary aptamer is not a simple target substance but a primary aptamer-target substance complex. Compared to using an aptamer pair that binds to two different binding sites of a target material for detection of a target material, according to the present invention, one aptamer recognizes a conjugate of another aptamer and a target material, so specificity and sensitivity can be greatly improved. .
상기 2차 압타머는 다음과 같이 제조할 수 있다. 먼저 특정 표적물질에 대하여 selex 방법을 통하여 1차 압타머를 선별한다. 이 때 표적물질과 특이적 결합을 하는 1차 압타머는 알려진 것을 이용할 수도 있다. 표적물질을 고정한 비드에 선별한 1차 압타머를 바인딩시킨 후 압타머 라이브러리를 통하여 이 결합체에 대해 selex round를 돌린 후 kd assay를 통해 가장 최적의 쌍을 찾아 내는 과정을 통해 2차 압타머를 선별한다. 따라서 본 발명의 2차 압타머는 단순 표적물질을 고정하여 선별한 1차 압타머와는 타겟, 염기서열, 3차원 구조 등의 측면에서 명백히 구분되는 것이다.The secondary aptamer can be prepared as follows. First, a primary aptamer is selected for a specific target material through the selex method. At this time, a known primary aptamer that specifically binds to the target material may be used. After binding the selected 1st aptamer to the beads immobilized with the target substance, a selex round is run on this conjugate through the aptamer library, and then the 2nd aptamer is selected through the process of finding the most optimal pair through kd assay do. Therefore, the secondary aptamer of the present invention is clearly distinguished from the primary aptamer selected by immobilizing a simple target material in terms of target, base sequence, and three-dimensional structure.
또한, 상기 2차 압타머는 도 8을 참조하여 다음과 같이 제조할 수 있다. 1차 압타머를 표면에 고정(plate, bead etc)하고 표적물질-1차 압타머 복합체를 형성한 상태에서 무작위 염기서열을 갖는 랜덤 라이브러리에 결합 반응을 유도한다. 이후 결합되지 않는 것들은 세척하여 제거하고 최종적으로 1차 압타머와 표적 복합체에 결합하는 압타머를 최종 선별한다.In addition, the secondary aptamer can be prepared as follows with reference to FIG. A primary aptamer is immobilized on a surface (plate, bead, etc.), and a binding reaction is induced to a random library having a random base sequence in a state in which a target material-primary aptamer complex is formed. Thereafter, those that do not bind are removed by washing, and finally, the aptamer that binds to the primary aptamer and the target complex is finally selected.
또한, 상기 2차 압타머는 도 8을 참조하여 다음과 같이 제조할 수 있다. 일반적인 SELEX 방법으로 선별된 Biotin 표지 1차 압타머를 streptavidin coated plate에 고정한다. 이후 결합하지 않는 것은 세척하여 제거하고 표적을 1차 압타머와 결합하여 1차 압타머-표적 복합체를 형성한다. 이후 2차 압타머를 선별하기 위해 무작위 라이브러리를 표적이 없는 1차 압타머가 고정된 plate well(1번 plate)에 처리하여 1차 압타머와 반응하는 라이브러리를 제거하고(counter selection(1)), 이를 His tag 등을 이용하여 표적물질을 고정한 plate에 반응시켜 표적물질에 반응하는 라이브러리를 제거한다(counter selection(2)). 여기에서 얻어진 라이브러리를 최종적으로 1차 압타머-표적 복합체와 반응하여 여기에 결합하는 2차 압타머를 선별할 수 있다. 상기 counter selection(1)과 (2)의 순서는 바뀔 수 있다.In addition, the secondary aptamer can be prepared as follows with reference to FIG. Biotin-labeled primary aptamers selected by the general SELEX method are immobilized on a streptavidin-coated plate. Thereafter, non-binding materials are removed by washing, and the target is combined with the primary aptamer to form a primary aptamer-target complex. Then, in order to select the secondary aptamer, the random library is treated in a plate well (plate No. 1) on which the primary aptamer without a target is immobilized to remove the library that reacts with the primary aptamer (counter selection (1)), This is reacted with a plate on which the target substance is immobilized using a His tag or the like to remove a library that reacts to the target substance (counter selection (2)). The library obtained here can finally react with the primary aptamer-target complex to select secondary aptamers that bind thereto. The order of the counter selection (1) and (2) may be changed.
도 1은 본 발명의 일 실시예에 따른 2차 압타머의 결합원리를 도식화한 것이다. 화살표 좌측은 표적물질(30)와 1차 압타머(10)가 결합한 표적물질-1차 압타머 복합체이며, 이 복합체에 대하여 친화성이 매우 높은 2차 압타머(20)가 특이적으로 결합하여 화살표 우측과 같은 복합체를 형성할 수 있다. 1 is a schematic diagram of the binding principle of a secondary aptamer according to an embodiment of the present invention. The left side of the arrow is a target substance-primary aptamer complex in which the target substance 30 and the primary aptamer 10 are bound, and the secondary aptamer 20 with very high affinity specifically binds to this complex. A complex can be formed as shown to the right of the arrow.
따라서 상기 2차 압타머는 상기 1차 압타머와 상기 표적물질이 결합함으로써 형성된 3차원 구조를 인식할 수 있다. 또한 2차 압타머는 1차 압타머의 적어도 일부 및 표적물질의 적어도 일부에 대하여 동시에 결합을 형성할 수 있다. 또한 2차 압타머는 표적물질과 1차 압타머가 결합함으로써 형성되는 1차 압타머의 구조 또는 표적물질의 구조 중 적어도 하나에 대하여 결합을 형성할 수 있다.Therefore, the secondary aptamer can recognize a three-dimensional structure formed by binding the primary aptamer and the target material. In addition, the secondary aptamer may simultaneously form a bond with at least a portion of the primary aptamer and at least a portion of the target material. In addition, the secondary aptamer may form a bond with at least one of the structure of the primary aptamer formed by binding the target material and the primary aptamer or the structure of the target material.
표적물질 또는 1차 압타머 단독으로 존재하는 경우에는 이들 각각에 대한 2차 압타머의 결합 친화성이 없거나 낮을 수 있다. 상기 2차 압타머는 상기 1차 압타머와 특이적 또는 비특이적 결합을 하지 않는 것일 수 있다. When the target material or the primary aptamer exists alone, the binding affinity of the secondary aptamer to each of them may be low or low. The secondary aptamer may not specifically or non-specifically bind with the primary aptamer.
또한, 상기 2차 압타머는 상기 1차 압타머-표적물질 복합체에 대한 결합 친화성이 상기 표적물질에 대한 결합 친화성에 대해 2 배 이상, 5 배 이상, 10 배 이상, 20 배 이상, 50 배 이상, 100 배 이상, 1000 배 이상, 또는 10000 배 이하, 5000 배 이하, 2000 배 이하, 1000 배 이하, 100 배 이하, 50 배 이하일 수 있고, 구체적으로는 2 내지 2000 배, 2 내지 1800 배, 또는 2 내지 1500 배, 2 내지 1000 배, 100 내지 1000 배, 2 내지 100 배, 10 내지 100 배, 2 내지 10 배, 또는 2 내지 5 배일 수 있다. In addition, the secondary aptamer has a binding affinity for the primary aptamer-target material complex that is 2-fold, 5-fold, 10-fold, 20-fold, or 50-fold higher than the binding affinity for the target material. , 100 times or more, 1000 times or more, or 10000 times or less, 5000 times or less, 2000 times or less, 1000 times or less, 100 times or less, 50 times or less, specifically 2 to 2000 times, 2 to 1800 times, or 2 to 1500 times, 2 to 1000 times, 100 to 1000 times, 2 to 100 times, 10 to 100 times, 2 to 10 times, or 2 to 5 times.
상기 결합 친화성은 상기 2차 압타머 말단에 측정 가능한 신호를 발생시킬 수 있는 물질로 표지한 후 신호의 세기를 비교함으로써 판단할 수 있다. 예컨대 2차 압타머 말단에 비오틴, HRP 등으로 표지한 후 형광값을 측정, 비교하여 결합 친화성을 비교할 수 있다.The binding affinity can be determined by labeling the end of the secondary aptamer with a substance capable of generating a measurable signal and then comparing the intensity of the signal. For example, binding affinity can be compared by labeling the end of the secondary aptamer with biotin, HRP, etc., and then measuring and comparing fluorescence values.
한편, 본 발명에 따른 압타머 복합체는, 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머; 및 상기 2차 압타머에 결합되어 표지하는 표지체;를 포함하는 것을 특징으로 한다.Meanwhile, the aptamer complex according to the present invention includes a primary aptamer formed by binding a target substance and a primary aptamer specifically binding to the target substance, and a secondary aptamer specifically binding to the target substance complex; and a label that binds to and labels the secondary aptamer.
또한, 상기 표지체는 형광체, 발광체, 양자점, 양자점 비드, 골드 나노 파티클, 유로피움, 염료, 방사성 표지, 전기화학적 작용기, 효소, 효소기질, 및 이들의 혼합물로 이루어진 군으로부터 선택될 수 있다.In addition, the marker may be selected from the group consisting of a fluorescent substance, a luminous substance, a quantum dot, a quantum dot bead, gold nanoparticles, europium, a dye, a radioactive label, an electrochemical functional group, an enzyme, an enzyme substrate, and mixtures thereof.
그리고, 상기 표지체는 양자점으로서, 상기 양자점은 12-16족계 화합물, 13-15족계 화합물일 수 있고, 양자점을 형성할 수 있는 반도체성 화합물을 포함할 수 있다. 상기 양자점은 코어-쉘 구조를 가질 수 있다. 상기 양자점은 카드뮴설파이드(CdS), 카드뮴셀레나이드(CdSe), 카드뮴텔레나이드(CdTe), 징크설파이드(ZnS), 징크셀레나이드(ZnSe), 징크텔레나이드(ZnTe), 징크옥사이드(ZnO), 징크옥사이드징크셀레나이드(ZnOZnSe), 징크옥사이드징크설파이드(ZnOZnS), 카드뮴옥사이드(CdO), 카드뮴셀레늄설파이드(CdSeS), 카드뮴셀레늄텔레나이드(CdSeTe), 카드뮴설파이드텔레나이드(CdSTe), 카드뮴징크설파이드(CdZnS), 카드뮴징크셀레나이드(CdZnSe), 카드뮴징크텔레나이드(CdZnTe), 징크셀레늄설파이드(ZnSeS), 징크셀레늄텔레나이드(ZnSeTe), 징크설파이드텔레나이드(ZnSTe), 카드뮴징크옥사이드(CdZnO), 징크셀레늄옥사이드(ZnSeO), 카드뮴셀레늄(CdSe), 카드뮴징크셀레늄설파이드(CdZnSeS), 카드뮴징크셀레늄텔레나이드(CdZnSeTe), 카드뮴셀레나이드징크설퍼(CdSeZnS), 카드뮴징크설파이드텔레나이드(CdZnSTe), 갈륨포스포러스(GaP), 갈륨아세나이드(GaAs), 갈륨니트라이드(GaN), 인듐포스포러스니트라이드(InPN), 인듐아세나이드니트라이드(InAsN), 인듐갈륨포스포러스(InGaP), 인듐갈륨아세나이드(InGaAs), 인듐갈륨니트라이드(InGaN), 인듐아세나이드니트라이드(InAsN), 인듐갈륨포스포러스(InGaP), 인듐알루미늄포스포러스안티모니(InAlPSb), 인듐갈륨포스포러스징크설파이드(InGaPZnS), 인듐갈륨포스포러스징크셀레나이드(InGaPZnSe), 인듐포스포러스징크셀레나이드(InPZnSe), 인듐포스포러스징크설파이드(InPZnS), 및 이들의 혼합물로 이루어진 군으로부터 선택될 수 있다.The label is a quantum dot, and the quantum dot may be a group 12-16 compound or a group 13-15 compound, and may include a semiconductor compound capable of forming quantum dots. The quantum dots may have a core-shell structure. The quantum dots are cadmium sulfide (CdS), cadmium selenide (CdSe), cadmium tellenide (CdTe), zinc sulfide (ZnS), zinc selenide (ZnSe), zinc tellenide (ZnTe), zinc oxide (ZnO), zinc Zinc oxide selenide (ZnOZnSe), zinc oxide zinc sulfide (ZnOZnS), cadmium oxide (CdO), cadmium selenium sulfide (CdSeS), cadmium selenium tellenide (CdSeTe), cadmium sulfide tellenide (CdSTe), cadmium zinc sulfide (CdZnS) ), cadmium zinc selenide (CdZnSe), cadmium zinc tellenide (CdZnTe), zinc selenium sulfide (ZnSeS), zinc selenium tellenide (ZnSeTe), zinc sulfide tellenide (ZnSTe), cadmium zinc oxide (CdZnO), zinc selenium Oxide (ZnSeO), Cadmium Selenium (CdSe), Cadmium Zinc Selenium Sulfide (CdZnSeS), Cadmium Zinc Selenium Tellenide (CdZnSeTe), Cadmium Selenide Zinc Sulfur (CdSeZnS), Cadmium Zinc Selenium Sulfide (CdZnSTe), Gallium Phosphorus ( GaP), gallium arsenide (GaAs), gallium nitride (GaN), indium phosphorus nitride (InPN), indium arsenide nitride (InAsN), indium gallium phosphorus (InGaP), indium gallium arsenide (InGaAs) , indium gallium nitride (InGaN), indium arsenide nitride (InAsN), indium gallium phosphorus (InGaP), indium aluminum phosphorus antimony (InAlPSb), indium gallium phosphorus zinc sulfide (InGaPZnS), indium gallium phosphorus It may be selected from the group consisting of zinc selenide (InGaPZnSe), indium phosphorus zinc selenide (InPZnSe), indium phosphorus zinc sulfide (InPZnS), and mixtures thereof.
그리고, 상기 표지체는 상기 2차 압타머와 공유결합, 이온결합, 또는 정전기적 결합을 통해 결합을 형성할 수 있다.In addition, the marker may form a bond with the secondary aptamer through a covalent bond, an ionic bond, or an electrostatic bond.
그리고, 상기 표지체는 상기 2차 압타머와 EDC 또는 EDC/NHS(또는 sulfo-NHS) 반응을 이용한 결합, 디설파이드 결합(S-S), 또는 말레이미드(maleimide)를 이용한 결합을 통해 결합을 형성할 수 있다.In addition, the marker may form a bond with the secondary aptamer through a bond using an EDC or EDC / NHS (or sulfo-NHS) reaction, a disulfide bond (S-S), or a bond using maleimide. have.
한편, 본 발명에 따른 진단키트는, 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머를 포함하는 것을 특징으로 한다.On the other hand, the diagnostic kit according to the present invention includes a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding a target substance and a primary aptamer that specifically binds to the target substance. characterized by
그리고, 상기 진단키트는 특이적 결합을 이용하는 진단키트일 수 있고 측면 유동 진단 스트립(LFA), 효소면역검출키트(ELISA), 바이오칩 등 표적물질을 검출하기 위한 공지의 검출장치일 수 있다.In addition, the diagnostic kit may be a diagnostic kit using specific binding, and may be a known detection device for detecting a target substance, such as a lateral flow diagnostic strip (LFA), an enzyme immunoassay kit (ELISA), or a biochip.
또한, 상기 진단키트는 표적물질과 특이적 결합을 하는 1차 압타머를 포함할 수 있다. In addition, the diagnostic kit may include a primary aptamer that specifically binds to a target substance.
또한, 상기 1차 압타머 및 2차 압타머 중 적어도 하나는 지지체에 고정된 것일 수 있다. 지지체는 진단키트의 내부면을 이루는 것을 의미할 수 있다.In addition, at least one of the primary aptamer and the secondary aptamer may be immobilized to the support. The support may mean forming the inner surface of the diagnostic kit.
또한, 상기 1차 압타머 및 2차 압타머는 비고정식일 수 있다. 이는 이들이 진단키트 내에서 자유롭게 이동할 수 있는 상태를 의미하는 것일 수 있다. 다만 표적물질 검출 과정에서 비고정 1차 압타머 또는 2차 압타머가 고정되는 단계를 가질 수 있다.In addition, the primary aptamer and the secondary aptamer may be non-fixed. This may mean a state in which they can freely move within the diagnostic kit. However, in the target material detection process, a step of immobilizing the non-immobilized primary aptamer or the secondary aptamer may be performed.
본 발명의 일 실시예에 따른 진단키트로서 측방유동센서(LFA)는 도 10과 같이, 시료투입패드 고정부(410), 반응패드 고정부(420), 검출패드(430) 및 흡수패드(440)가 구비되며 상기 시료투입패드 고정부(410)에 시료투입패드(411)를 고정하고, 상기 반응패드 고정부(420)에 반응패드(421)를 고정한 다음, 시료투입패드(411)에 시료액을 투입하면 시료액이 반응패드(421)를 거쳐 검출선(Test line)(431)과 대조선(Control line)(432)을 지나도록 이루어진 스트립을 사용한 측방유동센서(400)일 수 있다. As a diagnostic kit according to an embodiment of the present invention, the lateral flow sensor (LFA) includes a sample input pad fixing unit 410, a reaction pad fixing unit 420, a detection pad 430, and an absorption pad 440, as shown in FIG. ) is provided, the sample input pad 411 is fixed to the sample input pad fixing part 410, the reaction pad 421 is fixed to the reaction pad fixing part 420, and then the sample is placed on the sample input pad 411. It may be a lateral flow sensor 400 using a strip formed so that when the liquid is injected, the sample liquid passes through the reaction pad 421 and passes the test line 431 and the control line 432.
도 10의 상단 스트립은 표지체가 결합된 본 발명의 2차 압타머를 포함하는 반응패드와, 표적물질과 특이적 결합을 하는 1차 압타머가 고정된 검출선 및 2차 압타머와 결합하는 DNA가 고정된 대조선을 갖는 측방유동센서를 나타낸다. 시료가 투입되면 도 10의 하단 스트립과 같이 검출선에서 1차 압타머, 표적물질, 및 2차 압타머 복합체가 형성되어 시료 내 표적물질의 존재 여부 또는 농도에 대한 신호를 나타낼 수 있다. 특히 본 발명의 진단키트는 양자점 또는 양자점 비드를 표지체로 하였을 때 민감도를 현저히 향상시킬 수 있다. The upper strip of FIG. 10 shows a reaction pad containing the secondary aptamer of the present invention to which the marker is bound, a detection line to which the primary aptamer specifically binding to the target substance is immobilized, and DNA binding to the secondary aptamer. A lateral flow sensor with a fixed control line is shown. When a sample is introduced, a primary aptamer, a target substance, and a secondary aptamer complex are formed in the detection line as shown in the lower strip of FIG. In particular, the diagnostic kit of the present invention can significantly improve the sensitivity when using quantum dots or quantum dot beads as a marker.
한편, 본 발명에 따른 표적물질 검출방법은, 상기 본 발명에 따른 압타머를 이용하는 검출방법으로서, 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머를 이용하는 것을 특징으로 한다.On the other hand, the target substance detection method according to the present invention is a detection method using the aptamer according to the present invention, wherein the primary aptamer-target formed by the binding of the target substance and the primary aptamer that binds specifically to the target substance It is characterized by using a secondary aptamer that specifically binds to the material complex.
상기 표적물질 검출방법은 표적물질이 존재할 것으로 의심되는 시료를 상기 1차 압타머 및 상기 2차 압타머와 동시에 또는 시간차를 두고 접촉시키는 단계를 포함할 수 있다. The target material detection method may include contacting a sample suspected of having a target material with the primary aptamer and the secondary aptamer simultaneously or with a time difference.
상기 시료는 1차 압타머와 접촉한 후 2차 압타머와 접촉하거나, 2차 압타머와 접촉한 후 1차 압타머와 접촉하거나, 1차 압타머 및 2차 압타머와 동시에 접촉할 수 있다. 순서에 관계없이 시료 내 표적물질이 존재할 경우 표적물질이 1차 압타머와 만날 때 1차 압타머-표적물질 복합체가 형성되고, 반면에 시료 내 표적물질이 존재하지 않을 경우 1차 압타머-표적물질 복합체가 형성되지 않는다. 또한 순서에 관계없이 시료 내 표적물질이 존재할 경우 1차 압타머-표적물질 복합체가 2차 압타머에 노출되는 경우 1차 압타머-표적물질-2차 압타머 복합체를 형성하고, 반면에 시료 내 표적물질이 존재하지 않을 경우 1차 압타머-표적물질-2차 압타머 복합체가 형성되지 않는다.The sample may be contacted with a primary aptamer and then contacted with a secondary aptamer, contacted with a secondary aptamer and then contacted with a primary aptamer, or simultaneously contacted with a primary aptamer and a secondary aptamer. . Regardless of the order, if the target substance is present in the sample, when the target substance meets the primary aptamer, a primary aptamer-target substance complex is formed, whereas if the target substance does not exist in the sample, the primary aptamer-target substance is formed. No material complexes are formed. In addition, when the target substance is present in the sample regardless of the order, when the first aptamer-target substance complex is exposed to the second aptamer, a first aptamer-target substance-second aptamer complex is formed, whereas in the sample When the target substance does not exist, the primary aptamer-target substance-secondary aptamer complex is not formed.
상기 1차 압타머 및 2차 압타머 중 적어도 하나는 미리 지지체에 고정시킨 고정 압타머일 수 있다. 상기 1차 압타머 및 2차 압타머 중 하나가 고정된 경우 고정된 압타머를 고정 압타머 또는 포획 압타머라고 할 수 있고, 고정되지 않은 압타머를 비고정 압타머 또는 검출 압타머라고 할 수 있다. At least one of the primary aptamer and the secondary aptamer may be an immobilized aptamer previously immobilized on a support. When one of the primary aptamer and the secondary aptamer is immobilized, the immobilized aptamer may be referred to as an immobilized aptamer or a capture aptamer, and the non-immobilized aptamer may be referred to as a non-immobilized aptamer or a detection aptamer.
상기 1차 압타머 및 2차 압타머는 미리 지지체에 고정시키지 않은 비고정 압타머일 수 있다. 이 경우 표적물질이 존재할 것으로 의심되는 시료를 비고정 1차 압타머 및 비고정 2차 압타머와 동시에 또는 시간차를 두고 접촉시키는 단계; 및 상기 1차 압타머 및 상기 2차 압타머 중 적어도 하나가 지지체에 고정되는 단계;를 포함하는 검출방법에 의해 표적물질을 검출할 수도 있다.The primary aptamer and the secondary aptamer may be non-anchored aptamers that are not previously immobilized on a support. In this case, contacting a sample suspected of having a target substance with the non-immobilized primary aptamer and the non-immobilized secondary aptamer at the same time or with a time difference; and immobilizing at least one of the primary aptamer and the secondary aptamer to the support.
이와 관련하여, 상기 본 발명의 표적물질 검출방법을 일방향 유동이 진행되는 진단스트립 상에서 실시하는 경우 진단스트립 전단에 비고정 1차 압타머, 2차 압타머, 또는 이들의 혼합물을 준비하고, 시료를 주입하여 이들과 접촉시킨 후 유동에 의해 검출구역 등 특정 구역 도달 시 지지체와 상기 1차 압타머 또는 2차 압타머의 결합이 일어나도록 유도하는 방식으로 고정할 수도 있다. 이 때 지지체 표면과 고정이 일어나도록 하기 위해서 1차 압타머 및 2차 압타머 중 적어도 하나가 지지체와 결합가능한 작용기를 갖도록 개질된 것일 수 있다. 또한 지지체는 1차 압타머 및 2차 압타머 중 적어도 하나와 결합가능하도록 개질된 것일 수 있다. 이 때 압타머와 지지체 사이의 결합은 공유결합, 비공유 결합, 또는 정전기적 결합일 수 있다. 이에 따르면 시료 내 표적물질이 존재하는 경우 1차 압타머-표적물질-2차 압타머 복합체가 지지체에 고정될 수 있고, 시료 내 표적물질이 존재하지 않은 경우 지지체와 결합가능하도록 개질된 1차 압타머 또는 2차 압타머 단독 물질만이 고정될 것을 예상할 수 있다.In this regard, when the target material detection method of the present invention is carried out on a diagnostic strip in which one-way flow proceeds, a non-fixed primary aptamer, a secondary aptamer, or a mixture thereof is prepared at the front of the diagnostic strip, and the sample is After injecting and contacting them, it may be fixed in such a way as to induce binding between the support and the primary aptamer or the secondary aptamer when reaching a specific region such as a detection region by flow. At this time, in order to immobilize with the surface of the support, at least one of the primary aptamer and the secondary aptamer may be modified to have a functional group capable of binding to the support. In addition, the support may be modified to bind to at least one of the primary aptamer and the secondary aptamer. At this time, the bond between the aptamer and the support may be a covalent bond, a non-covalent bond, or an electrostatic bond. According to this, when the target substance is present in the sample, the first aptamer-target substance-secondary aptamer complex can be immobilized on the support, and when the target substance is not present in the sample, the modified primary pressure so as to bind to the support. One would expect that only tamers or secondary aptamers alone would be immobilized.
시료 내 표적물질이 존재한다고 가정했을 때 1차 압타머-표적물질-2차 압타머 복합체가 형성될 수 있도록 시료와 압타머들을 적절히 접촉시킨 후에는, 상기 1차 압타머-표적물질 복합체와 상기 2차 압타머가 결합하여 형성된 1차 압타머-표적물질-2차 압타머 복합체의 형성 여부를 판단하고, 이에 따라 시료 내 표적물질의 존부를 판단하는 단계를 가질 수 있다. 여기서 1차 압타머-표적물질-2차 압타머 복합체란 1차 압타머, 표적물질, 및 2차 압타머 사이에 형성된 결합으로 인해 복합체를 형성한 것을 의미하고, 이들 사이의 결합 구조를 한정하는 것은 아니다. Assuming that the target substance exists in the sample, after properly contacting the sample and the aptamers so that the first aptamer-target substance-secondary aptamer complex can be formed, the first aptamer-target substance complex and the second aptamer complex are formed. A step of determining whether a primary aptamer-target substance-secondary aptamer complex formed by binding of secondary aptamers is formed and, accordingly, determining the presence or absence of the target substance in the sample may be performed. Here, the primary aptamer-target material-secondary aptamer complex means that a complex is formed due to the bond formed between the primary aptamer, the target material, and the secondary aptamer, and the binding structure between them is defined. It is not.
상기 1차 압타머-표적물질-2차 압타머 복합체의 형성 여부는 PCR을 통해 판단할 수 있다. 구체적으로는, 먼저 마그네틱 비드에 타깃 물질을 컨쥬게이션 후 1차 압타머를 반응시킨다. 다음으로 원심분리로 미반응된 1차 압타머를 제거한 후 2차 압타머를 첨가하여 반응시킨다. 그 후 원심분리로 미반응된 2차 압타머를 제거한 후 비드에 바인딩된 2차 압타머를 인지하는 프라이머를 이용해 PCR로 확인할 수 있다. 이 때 비교군으로 타깃 물질이 없는 비드를 사용할 수 있다.Whether or not the first aptamer-target substance-secondary aptamer complex is formed can be determined through PCR. Specifically, first, after conjugating a target material to a magnetic bead, a primary aptamer is reacted. Next, after removing the unreacted primary aptamer by centrifugation, the secondary aptamer is added and reacted. Thereafter, unreacted secondary aptamers can be removed by centrifugation, and then PCR can be performed using primers recognizing the secondary aptamers bound to the beads. At this time, beads without a target material may be used as a comparison group.
또한, 상기 1차 압타머-표적물질-2차 압타머 복합체의 형성 여부는 시각적 신호를 통해 판단할 수 있다. 이 경우 상기 1차 압타머 또는 2차 압타머는 신호발생물질로 표지된 것일 수 있다. 그리고, 상기 1차 압타머 또는 2차 압타머는 양자점, 형광체, 발광체, 염색시약, 효소 염색(HRP) 및 이들의 혼합물로 이루어진 군으로부터 선택된 것으로 표지된 것일 수 있다. 상기 1차 압타머 또는 2차 압타머 중 어느 하나가 검출구역(지지체)에 고정되는 경우 고정되지 않은 이동상 압타머에 신호발생물질을 통해 표지하는 것이 표적물질 유무에 따라 검출구역에서의 신호발생에 차이가 발생할 수 있으므로 바람직하다.In addition, whether or not the first aptamer-target substance-secondary aptamer complex is formed can be determined through a visual signal. In this case, the primary aptamer or secondary aptamer may be labeled with a signal generating substance. In addition, the primary aptamer or secondary aptamer may be labeled with a material selected from the group consisting of quantum dots, fluorescent substances, luminescent substances, staining reagents, enzyme dyes (HRP), and mixtures thereof. When either of the primary aptamer or the secondary aptamer is immobilized in the detection zone (support), labeling the unimmobilized mobile aptamer with a signal generating material is associated with signal generation in the detection zone depending on the presence or absence of the target material. This is preferable because differences may occur.
[실시예][Example]
실시예 1: 2차 압타머의 선별Example 1: Selection of secondary aptamers
DEHP(Bis(2-ethylhexyl) phthalate (di-2-ethylhexyl phthalate, diethylhexyl phthalate)를 표적으로 하는 1차 압타머(HO-DEHP-57 (+), HO-DEHP-57 (-))에 대하여, 표적물질-1차 압타머 복합체를 표적으로 하는 2차 압타머 S008-A1-1 내지 S008-A1-9를 선별하였다. Regarding primary aptamers (HO-DEHP-57 (+), HO-DEHP-57 (-)) targeting DEHP (Bis (2-ethylhexyl) phthalate (di-2-ethylhexyl phthalate, diethylhexyl phthalate), Secondary aptamers S008-A1-1 to S008-A1-9 targeting the target substance-first aptamer complex were selected.
시험예 1: 1차 압타머와 2차 압타머의 비-특이 결합여부 확인Test Example 1: Confirmation of non-specific binding of primary aptamers and secondary aptamers
1차 압타머와 상기 실시예 1에서 선별한 2차 압타머의 염기서열 혼성화 등에 의한 비-특이 결합여부를 확인하기 위하여, 도 2와 같이 프로브(probe)와 1차 압타머의 복합체(100)가 고정되어 있는 플레이트(300)에 말단이 비오틴으로 표지된 2차 압타머(200)를 적용하였다.In order to confirm non-specific binding between the primary aptamer and the secondary aptamer selected in Example 1 by nucleotide sequence hybridization, etc., the complex 100 of the probe and the primary aptamer as shown in FIG. The secondary aptamer 200 labeled with biotin at the end was applied to the plate 300 on which is immobilized.
1차 압타머와 2차 압타머 사이의 결합여부를 확인하기 위해 streptavidin-HRP(Horseradish Peroxidase)를 결합하여 발광 값을 측정하였으며 형광측정값은 하기 표 1 및 도 3의 그래프와 같다. In order to confirm the binding between the primary aptamer and the secondary aptamer, streptavidin-HRP (Horseradish Peroxidase) was combined to measure the luminescence value, and the fluorescence measurement values are shown in the graphs of Table 1 and FIG. 3 below.
| 1차 압타머Primary aptamer | 2차 압타머Secondary aptamer |
형광측정값 (RLU: relative luminescence units)Fluorescence measurement (RLU: relative luminescence units) |
| HO-DEHP-57 (+)HO-DEHP-57 (+) | S008-A1-1(B31)S008-A1-1(B31) | 9315093150 |
| S008-A1-5(B32)S008-A1-5 (B32) | 3612036120 | |
| S008-A1-6(B33)S008-A1-6 (B33) | 29120002912000 | |
| S008-A1-9(B34)S008-A1-9 (B34) | 2723027230 | |
| HO-DEHP-57 (-)HO-DEHP-57 (-) | S008-A1-1(B31)S008-A1-1(B31) | 2784027840 |
| S008-A1-6(B33)S008-A1-6 (B33) | 2613026130 | |
| S008-A1-9(B34)S008-A1-9 (B34) | 108100108100 | |
| B-DEHP-57(positive control)B-DEHP-57 (positive control) | 3834000038340000 |
상기 형광측정 결과를 통해 1차 압타머와 2차 압타머 사이의 비특이 결합은 B33에서 아주 약하게 감지되는 것을 제외하고는 없는 것이 확인되었다.Through the fluorescence measurement results, it was confirmed that there was no non-specific binding between the primary aptamer and the secondary aptamer except that it was very weakly detected at B33.
시험예 2: 2차 압타머의 1차 압타머-표적물질 복합체에 대한 결합 친화성 확인Test Example 2: Confirmation of binding affinity of the secondary aptamer to the primary aptamer-target material complex
상기 실시예 1에서 선별한 2차 압타머의 1차 압타머-표적물질 복합체에 대한 결합 친화성을 확인하기 위하여 1차 압타머와 표적이 결합된 형태와 표적만 있는 형태에서 2차 압타머의 결합 정도를 확인하였다. In order to confirm the binding affinity of the secondary aptamer selected in Example 1 to the primary aptamer-target material complex, the primary aptamer and the target-bound form and the target-only form of the secondary aptamer The degree of binding was confirmed.
도 4와 같이, 1차 압타머(DEHP-57 Aptamer)(1차 압타머 농도 200 pmole/100μl, 2 μM)와, 표적물질인 DEHP 및 비드로 이루어지는 DEHP 비드(DEHP bead)를 접촉시켜 1차 압타머-표적물질 복합체의 형성을 유도하고, DEHP 2차 압타머(DEHP 2nd Aptamer)를 투입한 후 DEHP 2차 압타머 말단에 표지한 HRP를 통해 형광값을 측정하였다. As shown in FIG. 4, the primary aptamer (DEHP-57 Aptamer) (primary aptamer concentration: 200 pmole/100 μl, 2 μM) is brought into contact with the target material, DEHP, and DEHP beads composed of beads to form a primary aptamer. Formation of the aptamer-target material complex was induced, and after the introduction of DEHP 2nd Aptamer, the fluorescence value was measured through HRP labeled at the end of the DEHP 2nd aptamer.
이와 대조적으로, 1차 압타머가 존재하지 않는 경우(1차 압타머 농도 0 pmole)에 대하여 DEHP 비드와 DEHP 2차 압타머를 접촉시켜 마찬가지로 DEHP 2차 압타머 말단에 표지한 HRP를 통해 형광값을 측정하였다. In contrast, when the primary aptamer does not exist (primary aptamer concentration of 0 pmole), the DEHP bead and the DEHP secondary aptamer are brought into contact to measure fluorescence through HRP labeled at the end of the DEHP secondary aptamer. measured.
각 실험에 대하여 형광측정값은 하기 표 2 및 도 5의 그래프와 같다.Fluorescence measurement values for each experiment are shown in the graphs of Table 2 and FIG. 5 below.
| 2차 압타머 종류Types of secondary aptamers |
형광측정값 (RLU: relative luminescence units)Fluorescence measurement (RLU: relative luminescence units) |
|
| 1차 압타머 농도: 200 pmolePrimary aptamer concentration: 200 pmoles | 1차 압타머 농도: 0 pmolePrimary aptamer concentration: 0 pmole | |
| S008-A1-1S008-A1-1 | 202700202700 | 3459034590 |
| S008-A1-2S008-A1-2 | 3421000034210000 | 2325023250 |
| S008-A1-3S008-A1-3 | 3438034380 | 5144051440 |
| S008-A1-4S008-A1-4 | 264200264200 | 2423024230 |
| S008-A1-5S008-A1-5 | 2829000028290000 | 4097040970 |
| S008-A1-6S008-A1-6 | 1305000013050000 | 4175041750 |
| S008-A1-7S008-A1-7 | 6070060700 | 2435024350 |
| S008-A1-8S008-A1-8 | 174200174200 | 6046060460 |
| S008-A1-9S008-A1-9 | 202000000202000000 | 179900179900 |
| Library(negative control)Library (negative control) | 219100219100 | 5620056200 |
1차 압타머가 존재할 경우 그렇지 않은 경우에 비해 형광측정값이 현저하게 높았으며, 그렇지 않은 경우에는 모두 네거티브로 판단되는 경우였으며, 상기 형광측정값을 통해 2차 압타머는 1차 압타머와 표적이 결합한 상태에서만 복합체를 인지하여 결합하는 것을 확인하였다. In the presence of the primary aptamer, the fluorescence measurement value was significantly higher than in the case where it was not, and in all cases where it was not, it was judged as negative. It was confirmed that the complex was recognized and bound only in the state.
따라서 본 발명에 따른 2차 압타머는 1차 압타머 또는 표적물질에 대한 결합력이 매우 낮고 이에 비해 1차 압타머-표적물질 복합체에 대한 결합성이 현저히 높으므로 표적물질을 검출하는데 매우 효과적으로 사용될 수 있을 것이다.Therefore, the secondary aptamer according to the present invention has very low binding ability to the primary aptamer or target substance, and significantly higher binding to the primary aptamer-target substance complex, so it can be used very effectively for detecting the target substance. will be.
시험예 3: 표적물질 DEHP에 대한 샌드위치 어세이Test Example 3: Sandwich assay for target substance DEHP
표면의 BSA에 의해 1차 압타머(T17)를 표면에 고정시키고 1차 압타머의 Thiol기를 이용하여 BSA에 공유 결합시킨 다음, 표적물질인 DEHP를 적용하여 1차 압타머와 반응시키고, 비오틴 표지된 2차 압타머 B7, B30, B31, B32, 및 B33을 각각 적용하였다(도 6). The primary aptamer (T17) is immobilized on the surface by BSA on the surface and covalently bonded to BSA using the Thiol group of the primary aptamer, and then reacted with the primary aptamer by applying DEHP, a target material, and labeled with biotin. Secondary aptamers B7, B30, B31, B32, and B33 were applied, respectively (FIG. 6).
이에 대하여, 상기 2차 압타머가 1차 압타머-표적물질 복합체를 인지하여 결합한 정도를 streptavidin-HRP를 통해 측정하였으며, 그 결과는 도 7과 같았다. 1차 압타머인 T17번을 표면에 고정하고 2차 압타머인 B33으로 sandwich assay를 진행한 경우에 signal/noise 값(S/N ratio)이 3.5로 가장 높은 결과를 나타내었다.In contrast, the degree of recognition and binding of the secondary aptamer to the primary aptamer-target material complex was measured using streptavidin-HRP, and the results are shown in FIG. 7 . When the first aptamer, T17, was immobilized on the surface and the sandwich assay was performed with the second aptamer, B33, the highest signal/noise value (S/N ratio) was 3.5.
실시예 2: 1차 압타머와 표적 복합체에 결합하는 압타머 선별 - 2nd aptamer SELEXExample 2: Selection of aptamers that bind to the primary aptamer and the target complex - 2 nd aptamer SELEX
1차 압타머를 표면에 고정(plate, bead etc)하고 표적을 1차 압타머 복합체를 형성한 상태에서 무작위 염기서열을 갖는 랜덤 라이브러리에 결합 반응을 유도한다. 이후 결합되지 않는 것들은 세척하여 제거하고 최종적으로 1차 압타머와 표적 복합체에 결합하는 압타머를 최종 선별한다.A primary aptamer is immobilized on a surface (plate, bead, etc.), and a binding reaction is induced to a random library having a random base sequence in a state where a target is formed as a primary aptamer complex. Thereafter, those that do not bind are removed by washing, and finally, the aptamer that binds to the primary aptamer and the target complex is finally selected.
더욱 구체적으로는, 일반적인 SELEX 방법으로 선별된 Biotin 표지 1차 압타머를 streptavidin coated plate에 고정한다. 이후 결합하지 않는 것은 세척하여 제거하고 0.5 uM 농도의 COVID19 N protein을 1차 압타머와 결합하여 1차 압타머 표적 복합체를 형성한다. 이후 2차 압타머를 선별하기 위해 무작위 염기서열 N40을 갖는 라이브러리를 표적이 없는 1차 압타머 고정된 plate well(1번 plate)에 처리하여 1차 압타머와 반응하는 라이브러리를 제거하고 N protein의 His tag을 이용하여 고정한 plate에 1번 plate의 sup을 반응하여 N protein에 반응하는 라이브러리를 제거한다. 여기에서 얻어진 라이브러리를 최종적으로 1차 압타머 표적 복합체와 반응하여 여기에 결합하는 2차 압타머를 선별한다(도 8). 이를 통해 염기서열이 서로 다른 2nd 압타머 4 종(2nd Aptamer 1~4)을 선별하였다.More specifically, the Biotin-labeled primary aptamer selected by the general SELEX method is immobilized on a streptavidin-coated plate. After that, the unbound ones are removed by washing, and the COVID19 N protein at a concentration of 0.5 uM is combined with the primary aptamer to form the primary aptamer target complex. Then, in order to select the secondary aptamer, the library having the random base sequence N40 was treated with the target-free primary aptamer immobilized plate well (plate No. 1) to remove the library that reacts with the primary aptamer, and the N protein The library that reacts to N protein is removed by reacting sup of plate No. 1 on the fixed plate using His tag. The library obtained here is finally reacted with the primary aptamer target complex to select secondary aptamers that bind thereto (FIG. 8). Through this, four types of 2nd aptamers (2nd Aptamer 1-4) having different base sequences were selected.
시험예 4: 2차 압타머 결합력 검증Test Example 4: Verification of secondary aptamer binding force
라이브러리 2 종과 상기 실시예 2에서 선별한 2nd 압타머 4 종을 10 pmole/well 농도로 완충용액(40 mM HEPES pH 7.5, 102 mM NaCl, 5 mM KCl, 5 mM MgCl2 및 0.05 % Tween-20)에 희석하여 95 ℃에서 가열하고 상온까지 온도를 천천히 낮춰 구조를 안정화하였다.2 libraries and 4 2 nd aptamers selected in Example 2 were mixed at a concentration of 10 pmoles/well in a buffer solution (40 mM HEPES pH 7.5, 102 mM NaCl, 5 mM KCl, 5 mM MgCl 2 and 0.05% Tween- 20), heated at 95 °C, and slowly lowered the temperature to room temperature to stabilize the structure.
Biotin기가 결합된 aptamer를 표적 단백질과 혼합하여 결합반응을 진행하고 Dynal His-tag bead 10 ul를 첨가하여 RT에서 30 분간 결합반응하고 표적을 고정하였다. 이후 완충용액 100 ul로 3 회 세척하였다.The biotin-coupled aptamer was mixed with the target protein to proceed with the binding reaction, and 10 ul of Dynal His-tag bead was added to the binding reaction at RT for 30 minutes and the target was immobilized. After that, it was washed three times with 100 ul of the buffer solution.
1/2000 희석된 ST-HRP 100 ul를 각 well에 첨가하여 RT에서 30 min incubation 후, 완충용액 100 ul로 3 회 세척하였다.100 ul of 1/2000 diluted ST-HRP was added to each well, incubated at RT for 30 min, and washed three times with 100 ul of buffer solution.
Bio-rad Substrate A와 B를 섞어 100 ul씩 well에 첨가하여 ELISA 리더기로 발광을 측정하였으며(표 3 및 도 9), 4 종의 압타머 모두 결합력이 1 nM 안팎으로 측정되었다.Bio-rad Substrates A and B were mixed and added to wells by 100 ul, and luminescence was measured with an ELISA reader (Table 3 and FIG. 9), and the binding strength of all four aptamers was measured to be around 1 nM.
| N protein (nM)N protein (nM) | RLU(luminescence)Luminescence (RLU) | |||||
| Lib(1)Lib(1) | Lib(2)Lib(2) |
2nd Aptamer 12nd |
2nd Aptamer 22nd |
2nd Aptamer 32nd |
2nd Aptamer 42nd |
|
| 100100 | 428000428000 | 307400307400 | 2268300022683000 | 1066200010662000 | 5153900051539000 | 1468500014685000 |
| 2020 | 480000480000 | 827000827000 | 2482800024828000 | 1076400010764000 | 5555900055559000 | 1014000010140000 |
| 44 | 11160001116000 | 128000128000 | 2005800020058000 | 1154000011540000 | 4769900047699000 | 1082000010820000 |
| 0.80.8 | 991000991000 | 7100071000 | 1909800019098000 | 51630005163000 | 1436900014369000 | 55920005592000 |
| 0.160.16 | 460000460000 | 524000524000 | 41290004129000 | 29890002989000 | 30410003041000 | 14410001441000 |
| 0.0320.032 | 763000763000 | 142000142000 | 10540001054000 | 22240002224000 | 136000136000 | 305000305000 |
| 0.00640.0064 | 5100051000 | 342000342000 | 625000625000 | 814000814000 | 351000351000 | 652000652000 |
| 00 | 00 | 00 | 00 | 00 | 00 | 00 |
시험예 5: 측방유동센서의 제조 및 1차 압타머 2차 압타머 pair 선정을 위한 비교 평가Test Example 5: Preparation of lateral flow sensor and comparative evaluation for selection of primary aptamer secondary aptamer pair
도 10과 같이, 시료투입패드 고정부(410), 반응패드 고정부(420), 검출패드(430) 및 흡수패드(440)가 구비되며 상기 시료투입패드 고정부(410)에 시료투입패드(411)를 고정하고, 상기 반응패드 고정부(420)에 반응패드(421)를 고정한 다음, 시료투입패드(411)에 시료액을 투입하면 시료액이 반응패드(421)를 거쳐 검출선(Test line)(431)과 대조선(Control line)(432)을 지나도록 이루어진 스트립을 사용하여 진단키트인 측방유동센서(400)를 제조하였다.As shown in FIG. 10, a sample input pad fixing part 410, a reaction pad fixing part 420, a detection pad 430, and an absorption pad 440 are provided, and the sample input pad fixing part 410 has a sample input pad ( 411) is fixed, the reaction pad 421 is fixed to the reaction pad fixing part 420, and then the sample solution is injected into the sample input pad 411, and the sample solution passes through the reaction pad 421 to the detection line (Test). A lateral flow sensor 400, which is a diagnostic kit, was manufactured by using a strip formed to pass the line 431 and the control line 432.
양자점과 양자점 비드에 압타머를 conjugation하기 위하여 압타머의 5 prime에 thiol기를 접합하여 합성하였다. 양자점은 thiol-압타머를 혼합하여 16 시간 동안 반응 후 MWCO 100 kDa 튜브에 넣어 6,000 rpm에서 10 분간 원심분리하여 DI water를 이용하여 3 회 washing 하였고, 양자점 비드의 경우 양자점 비드에 Bovine serum albumin(BSA)를 정전기적 방법으로 결합 후 thiol-압타머를 혼합하여 BSA의 아미노산에 있는 thiol기와 S-S 결합이 되도록 하여 conjugation을 진행하였다. In order to conjugate the aptamer to the quantum dot and the quantum dot bead, it was synthesized by conjugating a thiol group to the 5 prime of the aptamer. Quantum dots were mixed with thiol-aptamer, reacted for 16 hours, put into a MWCO 100 kDa tube, centrifuged at 6,000 rpm for 10 minutes, and washed three times using DI water. In the case of quantum dot beads, Bovine serum albumin (BSA ) were combined by an electrostatic method, and thiol-aptamer was mixed to form an S-S bond with the thiol group in the amino acid of BSA to proceed with conjugation.
Control line은 1차 압타머의 3’에 cytosine(C) 10mer를 연장하고, 멤브레인의 컨트롤라인에는 Biotin-guanine(G) 10mer를 SA를 이용하여 고정하였다. 1차 압타머와 상기 실시예 2에서 선별한 3 종의 2차 압타머(2nd Aptamer 1~3)의 pair에 따라 타겟 단백질(N protein)의 첨가에 따른 테스트라인의 세기를 비교하여 압타머 pair를 선정하고자 하였다. 그 결과 도 11과 같이 멤브레인에 1차 압타머를 적용하고 2차 압타머 #3에 양자점을 표지한 pair에서 타겟 단백질에 의한 신호가 가장 우수한 것을 확인하였다.As the control line, cytosine (C) 10mer was extended to 3' of the primary aptamer, and Biotin-guanine (G) 10mer was fixed to the membrane control line using SA. According to the pair of the first aptamer and the three kinds of second aptamers (2nd Aptamer 1 to 3) selected in Example 2, the strength of the test line according to the addition of the target protein (N protein) was compared to compare the aptamer pair wanted to select. As a result, as shown in FIG. 11, it was confirmed that the signal by the target protein was the best in the pair in which the first aptamer was applied to the membrane and the second aptamer # 3 was labeled with quantum dots.
시험예 6: 금 나노입자와 양자점을 이용한 민감도 평가 비교Test Example 6: Comparison of sensitivity evaluation using gold nanoparticles and quantum dots
상기 시험예 5에 있어서, 양자점 또는 양자점 비드 대신 금 나노입자를 이용하여 측방유동센서를 제조한 후, 금 나노입자와 양자점을 이용한 진단키트의 민감도를 비교 평가하였다. 금 나노입자를 이용한 키트의 경우 N-protein 농도 별 테스트 결과 10 ug/mL농도까지 육안으로 확인되었다. 그에 비해 양자점 및 양자점 비드를 이용한 키트는 육안으로는 5 ug/mL 농도까지 확인이 가능하였으며, 형광 리더기를 이용하여 측정한 결과 1 ug/mL 농도까지 검출이 가능하였다(도 12 및 도 13).In Test Example 5, after manufacturing a lateral flow sensor using gold nanoparticles instead of quantum dots or quantum dot beads, the sensitivity of the diagnostic kit using gold nanoparticles and quantum dots was compared and evaluated. In the case of kits using gold nanoparticles, as a result of testing for each N-protein concentration, up to 10 ug/mL concentration was visually confirmed. In contrast, kits using quantum dots and quantum dot beads were able to visually confirm up to 5 ug/mL concentration, and as a result of measurement using a fluorescence reader, detection was possible up to 1 ug/mL concentration (FIGS. 12 and 13).
이상에서는 본 발명의 바람직한 실시예에 대해서 설명하였으나, 본 발명은 상술한 특정의 실시예에 한정되지 아니하며, 당해 기술분야에서 통상의 지식을 가진 자라면 본원 발명의 요지를 벗어남이 없이 다양한 변형 실시가 가능함은 물론이다. 따라서, 본 발명의 범위는 위의 실시예에 국한해서 해석되어서는 안되며, 후술하는 청구범위뿐만 아니라 이 청구범위와 균등한 것들에 의해 정해져야 할 것이다.Although the preferred embodiments of the present invention have been described above, the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications without departing from the gist of the present invention. Of course it is possible. Therefore, the scope of the present invention should not be construed as being limited to the above embodiments, and should be defined by not only the claims to be described later, but also those equivalent to these claims.
Claims (15)
- 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머인, 압타머.An aptamer, which is a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding a target substance and a primary aptamer that specifically binds to the target substance.
- 청구항 1에 있어서,The method of claim 1,상기 표적물질은 항원, 항체, 바이러스, 올리고뉴클레오티드, 단백질, 탄수화물, 과당, 글리코당, 올리고당, 호르몬, 수용체, 펩타이드, 기질, 대사물질, 전이상태 유사물, 보조인자, 저해제, 약물, 염료, 영양분, 성장인자, 조직, 무기 유기 영양소, 유해 환경물질, 또는 당단백질의 올리고당인 것을 특징으로 하는, 압타머.The target substance is an antigen, antibody, virus, oligonucleotide, protein, carbohydrate, fructose, glycosaccharide, oligosaccharide, hormone, receptor, peptide, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient , Growth factors, tissues, inorganic organic nutrients, harmful environmental substances, or oligosaccharides of glycoproteins, characterized in that, aptamers.
- 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머; 및a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding a target substance and a primary aptamer that specifically binds to the target substance; and상기 2차 압타머에 결합되어 표지하는 표지체;를 포함하는, 압타머 복합체.Containing, an aptamer complex that binds to and labels the secondary aptamer.
- 청구항 3에 있어서,The method of claim 3,상기 표지체는 형광체, 발광체, 양자점, 양자점 비드, 골드 나노 파티클, 유로피움, 염료, 방사성 표지, 전기화학적 작용기, 효소, 효소기질, 및 이들의 혼합물로 이루어진 군으로부터 선택되는 것을 특징으로 하는, 압타머 복합체.The label is selected from the group consisting of a phosphor, a luminous body, a quantum dot, a quantum dot bead, gold nanoparticles, europium, a dye, a radioactive label, an electrochemical functional group, an enzyme, an enzyme substrate, and a mixture thereof, tamer complex.
- 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머를 포함하는 진단키트.A diagnostic kit comprising a target substance and a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding of a primary aptamer that specifically binds to the target substance.
- 청구항 5에 있어서,The method of claim 5,상기 표적물질과 특이적 결합을 하는 1차 압타머를 포함하는 것을 특징으로 하는, 진단키트.A diagnostic kit comprising a primary aptamer that specifically binds to the target substance.
- 청구항 6에 있어서,The method of claim 6,상기 1차 압타머 및 2차 압타머 중 적어도 하나는 지지체에 고정된 것을 특징으로 하는, 진단키트.The diagnostic kit, characterized in that at least one of the primary aptamer and the secondary aptamer is immobilized on the support.
- 청구항 6에 있어서,The method of claim 6,상기 1차 압타머 및 2차 압타머는 비고정식인 것을 특징으로 하는, 진단키트.The diagnostic kit, characterized in that the primary aptamer and the secondary aptamer are non-fixed.
- 표적물질 및 상기 표적물질과 특이적 결합을 하는 1차 압타머가 결합하여 형성된 1차 압타머-표적물질 복합체와 특이적 결합을 하는 2차 압타머를 이용한 표적물질 검출방법.A method for detecting a target substance using a secondary aptamer that specifically binds to a primary aptamer-target substance complex formed by binding a target substance and a primary aptamer that specifically binds to the target substance.
- 청구항 9에 있어서,The method of claim 9,표적물질이 존재할 것으로 의심되는 시료를 상기 1차 압타머 및 상기 2차 압타머와 동시에 또는 시간차를 두고 접촉시키는 단계를 포함하는, 표적물질 검출방법.A target material detection method comprising the step of contacting a sample suspected of having a target material with the primary aptamer and the secondary aptamer simultaneously or with a time difference.
- 청구항 10에 있어서,The method of claim 10,상기 1차 압타머 및 2차 압타머 중 적어도 하나는 지지체에 고정된 것을 특징으로 하는, 표적물질 검출방법.Characterized in that at least one of the primary aptamer and the secondary aptamer is immobilized to the support, the target material detection method.
- 청구항 9에 있어서,The method of claim 9,표적물질이 존재할 것으로 의심되는 시료를 비고정 1차 압타머 및 비고정 2차 압타머와 동시에 또는 시간차를 두고 접촉시키는 단계; 및contacting a sample suspected of having a target substance with the non-anchored primary aptamer and the non-anchored secondary aptamer at the same time or with a time difference; and상기 1차 압타머 및 상기 2차 압타머 중 적어도 하나가 지지체에 고정되는 단계;를 포함하는, 표적물질 검출방법.A method for detecting a target material, comprising: fixing at least one of the primary aptamer and the secondary aptamer to a support.
- 청구항 9에 있어서,The method of claim 9,상기 1차 압타머-표적물질 복합체와 상기 2차 압타머가 결합하여 형성된 1차 압타머-표적물질-2차 압타머 복합체의 형성 여부에 따라 시료 내 표적물질의 존부를 판단하는 단계를 포함하는, 표적물질 검출방법.Determining whether a target substance is present in a sample according to whether a first aptamer-target substance-secondary aptamer complex formed by binding the first aptamer-target substance complex and the second aptamer is formed, Target substance detection method.
- 청구항 13에 있어서, The method of claim 13,상기 1차 압타머-표적물질-2차 압타머 복합체의 형성 여부는 PCR을 통해 판단하는 것을 특징으로 하는, 표적물질 검출방법.The first aptamer-target material-target material detection method, characterized in that the formation of the secondary aptamer complex is determined by PCR.
- 청구항 13에 있어서,The method of claim 13,상기 1차 압타머-표적물질-2차 압타머 복합체의 형성 여부는 시각적 신호를 통해 판단하는 것을 특징으로 하는, 표적물질 검출방법.The target substance detection method, characterized in that the formation of the first aptamer-target substance-secondary aptamer complex is determined through a visual signal.
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