KR20170099611A - Dual aptamer for detecting Blood coagulation factor Ⅱa and use thereof - Google Patents
Dual aptamer for detecting Blood coagulation factor Ⅱa and use thereof Download PDFInfo
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Abstract
One example of the present invention is a dual appamer for the detection of blood coagulation factor < RTI ID = 0.0 > IIa < / RTI > characterized in that the aptamer for blood coagulation factor IIa and the aptamer for hemin are present in bound form via linker molecules in the form of oligonucleotide Lt; / RTI > In the case of using the dual-app tamer according to one embodiment of the present invention, the blood coagulation factor IIa present in the blood sample can be detected quickly and accurately quantitatively. Therefore, by using the method for detecting blood coagulation factor IIa or the kit for detecting blood coagulation factor IIa according to an example of the present invention, it is possible to quickly diagnose a patient who develops blood clots or bleeding, and take necessary measures promptly .
Description
The present invention relates to a dual appamer for detecting blood coagulation factors and uses thereof, and more particularly to a dual appamer for detecting blood coagulation factors capable of competitively binding to blood coagulation factors and hemin, A method capable of quantitatively detecting a coagulation factor or a kit capable of quantitatively detecting blood coagulation factors.
Blood coagulation is a hemostatic device that coagulates by changing fibrinogen into fibrin in the blood serum. Fig. 1 schematically shows the blood coagulation reaction mechanism. As shown in FIG. 1, the blood coagulation reaction includes blood coagulation factor I (commonly known as fibrinogen), Ⅱ (commonly known as prothrombin), Ⅲ (commonly known as tissue thromboplastin), Ⅳ (commonly known as calcium ion) (Common name: Stuart-Plough factor), XI (common name: plasma thromboplastin), Ⅶ (common name: procombellulin), Ⅶ , And XII (commonly known as the causal agent), are involved in various blood coagulation factors.
On the other hand, fibrin produced by the blood clotting reaction becomes the main component of the thrombus. Thrombosis refers to a mass of blood clotted locally in the blood vessel or heart. The disease caused by such thrombosis is called thrombosis, and it is also referred to as thromboembolism. If the thrombosis gets worse, there will be various obstacles in the human body and death due to myocardial infarction and cerebral palsy. A variety of anticoagulants have been developed to prevent or treat these thromboses. Conventional anti-blood coagulants such as Wafarin target several blood coagulation factors, while recent anti-blood coagulants target single blood coagulation factors. For example, anti-coagulants such as Rivaroxaban, Apixaban and Edoxaban are targeted to the active form of blood coagulation factor X, Xa, and Dabigatran, Targets the active type Ⅱa of blood coagulation factor II (commonly known as thrombin).
Patients with thrombosis usually take anticoagulants once a day or twice a day, depending on the doctor's instructions. For example, if a patient with thrombosis increases the frequency of administration of an anticoagulant or decreases the dose, the level of blood coagulation factors in the body may become too high, causing the thrombosis to become more severe and myocardial infarction and the like may occur. In addition, when a patient with thrombosis reduces the administration period of the anti-coagulant or increases the dose, the blood coagulation factor level in the human body becomes too low, thereby causing excessive bleeding. Therefore, the blood coagulation factor level in the human body should be maintained in an appropriate range. To monitor whether the level of the coagulation factor in the human body is maintained in an appropriate range, the blood coagulation factor IIa or the blood concentration of the coagulation factor Should be quantitatively detected.
With regard to the detection of blood coagulation factors, Korean Patent Laid-Open Publication No. 10-2008-0025043 discloses a method of contacting a layer of blood sample with a fluorogenic substrate of thrombin, said layer having a thickness in the range of 0.05 mm to 5 mm And a surface within the range of 10 mm < 2 > to 500 mm < 2 >; Producing thrombin in the sample; And measuring the fluorescence emitted from the surface of the layer by a fluorophore released from the fluorogenic substrate as a result of the enzymatic action of the generated thrombin on the fluorescent substrate, wherein the thrombin activity in the sample is measured in vitro A method of measuring the temperature of the sample is disclosed. Korean Patent Registration No. 10-1113793 discloses a microcantilever sensor; A DNA compactor fixed on the microcantilever; A measuring device for measuring a resonant frequency of the micro cantilever; And a spacer for backfilling, wherein the DNA aptamer comprises a 5'-GGT TGG TGT GGT TGG-3 'sequence, wherein the DNA aptamer-guard-DNA The present invention relates to a microcantilever sensor-based thrombin detection device. Korean Patent Laid-Open Publication No. 10-2015-0142723 discloses a method for manufacturing an ITO electrode, comprising the steps of: preparing a surface of an ITO electrode having a hydroxy group bonded thereto by treating distilled water on the surface of an ITO electrode treated with a cleaning solution; And a second step of dropping and coating the fibrinogen solution on the surface of the ITO electrode prepared in the first step. Korean Patent Laid-Open Publication No. 10-2011-0126940 discloses a thrombin detection sensor in which an aptamer that is selectively bound to thrombin is fixed on an electrode surface and nanoparticles coated with an electron transfer mediator are bonded to the fixed abutment sensor .
SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel double appamer which is used for quantitatively and reliably detecting a minute amount of blood coagulation factor IIa present in a blood sample. It is also an object of the present invention to provide a method for quantitatively and reliably detecting a trace amount of blood coagulation factor IIa present in a blood sample. It is also an object of the present invention to provide a kit capable of quantitatively and reliably detecting a trace amount of blood coagulation factor IIa present in a blood sample.
It is another object of the present invention to provide a novel dual appamer used for quantitatively and reliably detecting a trace amount of blood coagulation factor Xa present in a blood sample. Another object of the present invention is to provide a method for quantitatively and reliably detecting a trace amount of blood coagulation factor Xa present in a blood sample. Another object of the present invention is to provide a kit capable of quantitatively and reliably detecting a trace amount of blood coagulation factor Xa present in a blood sample.
The inventors of the present invention have found that a novel double aptamer capable of simultaneously binding with blood coagulation factors and hemin can be produced by reacting a blood sample with a blood sample in the presence of a predetermined concentration of hemin, The present inventors have confirmed that a trace amount of blood coagulation factors can be quantitatively and reliably detected, thereby completing the present invention.
In order to solve one object of the present invention, an example of the present invention is characterized in that the aptamer for the blood coagulation factor IIa and the aptamer for the hemin exist in a form bound through an oligonucleotide-type linker molecule Lt; RTI ID = 0.0 > IIa < / RTI >
In order to solve one object of the present invention, an example of the present invention is a method for detecting a blood coagulation factor IIa comprising the steps of adding a blood sample to a solution containing hematoxylin and hemin, ; Adding a chromogenic substrate and hydrogen peroxide to the first reaction mixture and reacting to form a second reaction mixture; And measuring the chromaticity or absorbance of the second reaction mixture.
In order to solve one object of the present invention, an example of the present invention is a method for detecting a blood coagulation factor IIa comprising the steps of adding a blood sample to a solution containing hematoxylin and hemin, ; Adding a chromogenic substrate and hydrogen peroxide to the first reaction mixture and reacting to form a second reaction mixture; Adding a luminous inducer and hydrogen peroxide to the second reaction mixture and reacting to form a third reaction mixture; And measuring the luminescence intensity of the third reaction mixture.
In order to solve one object of the present invention, one example of the present invention relates to a method for producing a blood coagulation factor IIa, comprising the steps of: preparing a blood coagulation factor IIa-detecting solution containing a double aptamer-containing solution, a solution containing a hemin, a solution containing a chromogenic substrate and a solution containing a hydrogen peroxide And provides a kit for detecting factor IIa.
In order to solve one object of the present invention, one example of the present invention is a solution containing a double aptamer for detecting blood coagulation factor IIa, a solution containing hemin, a solution containing a chromogenic substrate, a solution containing a luminescent inducer and a solution containing hydrogen peroxide The present invention also provides a kit for detecting blood coagulation factor IIa.
In order to solve the other object of the present invention, another example of the present invention is characterized in that the aptamer for the blood coagulation factor Xa and the aptamer for the hemin exist in a form bound through the linker molecule in the form of oligonucleotide Lt; / RTI > for the detection of blood coagulation factor Xa.
In order to solve the other object of the present invention, another example of the present invention is a method for detecting a blood coagulation factor Xa, comprising the steps of adding a blood sample to a solution containing double aptamer for detecting coagulation factor Xa and hemin, ; Adding a chromogenic substrate and hydrogen peroxide to the first reaction mixture and reacting to form a second reaction mixture; And measuring the chromaticity or absorbance of the second reaction mixture.
In order to solve the other object of the present invention, another example of the present invention is a method for detecting a blood coagulation factor Xa, comprising the steps of adding a blood sample to a solution containing double aptamer for detecting coagulation factor Xa and hemin, ; Adding a chromogenic substrate and hydrogen peroxide to the first reaction mixture and reacting to form a second reaction mixture; Adding a luminous inducer and hydrogen peroxide to the second reaction mixture and reacting to form a third reaction mixture; And measuring the luminescence intensity of the third reaction mixture. The present invention also provides a method for detecting blood coagulation factor Xa.
In order to solve the other object of the present invention, another example of the present invention is a blood coagulation agent comprising the double-aptamer-containing solution for detecting blood coagulation factor Xa, the solution containing hemin, the solution containing chromogenic substrate and the solution containing hydrogen peroxide And provides a kit for detecting factor Xa.
In order to solve the other object of the present invention, another example of the present invention is a solution containing a double aptamer-containing solution for detection of blood coagulation factor Xa, a solution containing hemin, a solution containing a chromogenic substrate, a solution containing a luminescent inducer and a solution containing hydrogen peroxide A kit for detecting blood coagulation factor Xa comprising a solution is provided.
In the case of using the double-aptamer according to one embodiment of the present invention, the blood coagulation factor IIa present in the blood sample can be detected quickly and accurately quantitatively. Therefore, by using the method for detecting blood coagulation factor IIa or the kit for detecting blood coagulation factor IIa according to an example of the present invention, it is possible to quickly diagnose a patient who develops blood clots or bleeding, and take necessary measures promptly .
Fig. 1 schematically shows the blood coagulation reaction mechanism.
FIG. 2 schematically shows a method for detecting blood coagulation factor IIa according to a preferred embodiment of the present invention.
FIG. 3 schematically shows a method for detecting blood coagulation factor Xa according to another preferred embodiment of the present invention.
FIG. 4 is a graph showing the relationship between the concentration of blood coagulation factor IIa and the intensity of chemiluminescence, which are present in the assay sample, on the basis of the result of the measurement of the reactivity of the double aptamer No. 3 to blood coagulation factor IIa by the method of Test Example 2 It is a graph.
FIG. 5 is a graph showing the relationship between the concentration of blood coagulation factor IIa present in the assay sample and the reciprocal of the chemiluminescence intensity of the assay sample 2, after measuring the reactivity of the double aptamer No. 3 to the blood coagulation factor IIa by the method of Test Example 2 This is the plotted graph.
Fig. 6 is a graph showing the results obtained by the method of Test Example 3; 7 < / RTI > for blood coagulation factor Xa.
FIG. 7 is a graph showing the relationship between the concentration of blood coagulation factor Xa present in the assay sample and the chemiluminescence intensity of the assay sample, based on the result of measuring the reactivity of the double-aptamer No. 7 to blood coagulation factor Xa by the method of Test Example 4. FIG. It is a graph.
FIG. 8 is a graph showing the relationship between the concentration of blood coagulation factor Xa present in the assay sample and the reciprocal of the chemiluminescence intensity based on the result of the measurement of the reactivity of the double-aptamer No. 7 to blood coagulation factor Xa by the method of Test Example 4 This is the plotted graph.
Hereinafter, terms used in the present invention will be described.
As used herein, the term " blood coagulation factor IIa "refers to the active form of blood coagulation factor II (commonly known as prothrombin), which represents thrombin.
As used herein, the term " blood coagulation factor Xa "refers to the active form of the blood coagulation factor X (commonly known as Stuart-Prower factor).
As used herein, the term "aptamer" refers to a single or double-stranded nucleic acid or oligonucleotide in the form of an oligonucleotide capable of binding with high affinity and specificity to a target molecule such as a protein, .
As used herein, the term "DNA aptamer" refers to an aptamer consisting of oligonucleotides in the form of DNA or modified DNA.
As used herein, the term "RNA aptamer" refers to an aptamer consisting of an RNA form or an oligonucleotide of modified RNA form.
Hereinafter, the present invention will be described in detail.
One aspect of the present invention relates to a novel dual appamer used to quantitatively and reliably detect a trace amount of blood coagulation factor IIa present in a blood sample. A novel double aptamer for detecting blood coagulation factor IIa according to an example of the present invention exists in a form in which aptamer for hemocyte factor IIa and aptamer for hemin are bound through linker molecules in the form of oligonucleotide. The aptamer for the blood coagulation factor IIa may be selected from aptamers for various known blood coagulation factors IIa or aptamers for novel blood coagulation factors IIa, preferably DNA aptamers. In addition, the aptamer for the hemin may be selected from aptamer for various known hemin or an aptamer for new hemin, preferably a DNA aptamer. The linker molecule may also be selected from oligonucleotides capable of binding to aptamers for blood coagulation factor IIa and aptamers for hemin via a phosphodiester bond, preferably 3 < RTI ID = 0.0 > To 10 consecutive nucleotides.
The novel double aptamer for blood coagulation factor IIa detection according to an example of the present invention can be represented by the following
[Formula 1]
5'-P IIa- LQ H -3 '
[Formula 2]
5'-Q H -LP IIa -3 '
In the
The novel double aptamer for detecting blood coagulation factor IIa represented by the
The novel dual aptamer for blood coagulation factor IIa detection according to an embodiment of the present invention competitively binds to blood coagulation factor IIa and hemin and when the aptamer part for blood coagulation factor IIa binds to blood coagulation factor IIa The aptamer moiety for hemin is less responsive to hemin. In addition, when hemin binds to the aptamer portion of hemin, a DNA-based enzyme in the form of a quadruplex structure is formed. Since the DNA-based enzyme has a peroxidase activity, it can replace horseradish peroxidase and the like. On the other hand, when the blood coagulation factor IIa is present in a relatively larger amount than the hemin in the assay sample, the novel double aptamer for detecting coagulation factor IIa according to the present invention exhibits a weak peroxidase activity, When the blood coagulation factor IIa is present in the assay sample in a relatively small amount compared to hemin, the novel dual aptamer for detecting coagulation factor IIa according to an embodiment of the present invention exhibits a strong peroxidase activity. The blood coagulation factor IIa present in the blood sample can be quantitatively detected using the above characteristics of the novel dual appamer for detecting coagulation factor IIa according to an example of the present invention.
One aspect of the present invention relates to a method for quantitatively and reliably detecting a trace amount of blood coagulation factor IIa present in a blood sample. The method for detecting blood coagulation factor IIa according to an embodiment of the present invention comprises the steps of adding a blood sample to a solution containing both double aptamer for detection of blood coagulation factor IIa and hemin and reacting to form a first reaction mixture ; Adding a chromogenic substrate and hydrogen peroxide to the first reaction mixture and reacting to form a second reaction mixture; And measuring the chromaticity or absorbance of the second reaction mixture. The coloring substrate is not limited in its kind as long as it can be converted into a predetermined coloring substance by reacting with hydrogen peroxide in the presence of a DNA-based enzyme having peroxidase or peroxidase activity, For example, TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], luminol, isoluminol ), Amplex-Red (10-acetyl-3,7-dihydroxyphenoxazine) or Amplex-UltraRed, and Amplex-Red (10-acetyl-3,7-dihydroxyphenoxazine) or Amplex-UltraRed. Amplex-Red (10-acetyl-3,7-dihydroxyphenoxazine) or Amplex-UltraRed can be used in a 1: 1 stoichiometric ratio with hydrogen peroxide in the presence of DNA-based enzymes (peroxidase or peroxidase) To generate a red fluorescent material, resorufin.
A method for detecting blood coagulation factor IIa according to a preferred embodiment of the present invention is characterized in that a blood sample is added to a solution containing both of a dual appamer for detection of blood coagulation factor IIa and hemin, ; Adding a chromogenic substrate and hydrogen peroxide to the first reaction mixture and reacting to form a second reaction mixture; Adding a luminous inducer and hydrogen peroxide to the second reaction mixture and reacting to form a third reaction mixture; And measuring the emission intensity of the third reaction mixture. In the method of detecting blood coagulation factor IIa according to a preferred embodiment of the present invention, the chromogenic substrate reacts with hydrogen peroxide in the presence of a DNA-based enzyme exhibiting peroxidase or peroxidase activity, For example, TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline 6-sulfonic acid], luminol, isoluminol, 10-acetyl-3,7-dihydroxyphenoxazine or Amplex-UltraRed, and Amplex-Red (10-acetyl -3,7-dihydroxyphenoxazine) or Amplex-UltraRed, and it is preferably Amplex-Red (10-acetyl-3,7-dihydroxyphenoxazine) or Amplex-UltraRed. Amplex-Red (10-acetyl-3,7-dihydroxyphenoxazine) or Amplex-UltraRed can be used in a 1: 1 stoichiometric ratio with hydrogen peroxide in the presence of DNA-based enzymes (peroxidase or peroxidase) To generate a red fluorescent material, resorufin. In addition, the luminescent inducing material reacts with hydrogen peroxide to generate a high-energy intermediate material, and through the high-energy intermediate material, energy is transferred to a chromogenic fluorescent material, which is a product of the chromogenic substrate. Ultimately, The type of the chemiluminescence is not limited as long as it releases chemiluminescence. For example, ODI (1,1'-oxalyldiimidazole), OD2MI (1,1'-oxalyldi-2-methyl- imidazole) 1-oxalyldi-4-methyl-imidazole), OD2EI (1,1'-oxalyldi-2-ethyl- imidazole), OD4EI (1,1'- 1'-oxalyldisodium benzoate, OD2B (1,1'-oxalyldi-2-sodium benzoate) or OD4B (1,1'-oxalyldi-4-sodium benzoate). The present invention refers to both the disclosures of U.S. Patent Application Publication No. 2004/0142358 and U.S. Patent No. 8,492,101 concerning chemiluminescence methods using ODI, ODI derivatives, ODB or ODB derivatives. FIG. 2 schematically shows a method for detecting blood coagulation factor IIa according to a preferred embodiment of the present invention. As shown in FIG. 2, when a double-appamer and hemine for detecting blood coagulation factor IIa are put into blood or plasma in which blood coagulation factor IIa is present, blood coagulation factor IIa and hemin are competitively bound to a double appetamer for detection of blood coagulation factor IIa . At this time, when the blood coagulation factor IIa is bound to the aptamer portion of the blood coagulation factor IIa, which constitutes a double appetamer for detecting the coagulation factor Ⅱa, the reactivity between the aptamer moiety for hemin and hemin is considerably reduced. On the other hand, hemin associates with the aptamer portion of the hemin to form a quadruple-shaped DNAzyme. The DNAzyme has an enzyme activity such as HRP (Horseradish peroxidase). In addition, Amplex Red is converted to resorufin, which can cause strong chemiluminescence in the presence of peroxidase such as HRP and hydrogen peroxide. Thus, Amplex Red is converted to resorufin in the presence of quadruplicate DNAzymes and hydrogen peroxide. And resorufin releases red light in the presence of ODI (1,1'-oxalyldiimidazole) and hydrogen peroxide.
One aspect of the present invention relates to a kit capable of quantitatively and reliably detecting a trace amount of blood coagulation factor IIa present in a blood sample. A kit for detecting blood coagulation factor IIa according to an embodiment of the present invention comprises a double aptamer-containing solution, a hemin-containing solution, a chromogenic substrate-containing solution and a hydrogen peroxide-containing solution for detecting blood coagulation factor IIa described above. The method for detecting blood coagulation factor IIa according to an embodiment of the present invention can be implemented using a kit for detecting blood coagulation factor IIa according to an example of the present invention. In addition, a kit for detecting blood coagulation factor IIa according to a preferred embodiment of the present invention is a kit for detecting blood coagulation factor IIa, comprising a solution containing a double aptamer for detecting blood coagulation factor IIa, a solution containing hemin, a solution containing chromogenic substrate, And hydrogen peroxide-containing solution. The method for detecting blood coagulation factor IIa according to a preferred embodiment of the present invention can be implemented using a kit for detecting blood coagulation factor IIa according to a preferred embodiment of the present invention. In the kit for the detection of blood coagulation factor IIa of the present invention, the chromogenic substrate is preferably TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline- 6-sulfonic acid], luminol, isoluminol, Amplex-Red (10-acetyl-3,7-dihydroxyphenoxazine) or Amplex-UltraRed. The light emitting inducing material is preferably selected from the group consisting of 1,1'-oxalyldiimidazole (ODI), 1,1'-oxalyldi-2-methyl-imidazole (OD2MI), and 1,1'-oxalyldi- ), OD2EI (1,1'-oxalyldi-2-ethyl-imidazole), OD4EI (1,1'-oxalyldi-4-ethyl- imidazole), ODB (1,1'-oxalyldisodium benzoate) '-oxalyldi-2-sodium benzoate) or OD4B (1,1'-oxalyldi-4-sodium benzoate).
Another aspect of the present invention relates to a novel dual appamer used to quantitatively and reliably detect a trace amount of blood coagulation factor Xa present in a blood sample. In another example of the present invention, a novel dual aptamer for the detection of coagulation factor Xa is present in a form in which the aptamer for blood coagulation factor Xa and the aptamer for hemin are bound through linker molecules in the form of oligonucleotides. The aptamer for the blood coagulation factor Xa may be selected from aptamers for a variety of known blood coagulation factors Xa or aptamers for novel blood coagulation factors Xa, preferably RNA aptamers. In addition, the aptamer for the hemin may be selected from aptamer for various known hemin or an aptamer for new hemin, preferably a DNA aptamer. The linker molecule may also be selected from oligonucleotides capable of binding to aptamers for blood coagulation factor IIa and aptamers for hemin via a phosphodiester bond, preferably 3 < RTI ID = 0.0 > To 10 consecutive nucleotides.
The novel double aptamer for detecting blood coagulation factor Xa according to another embodiment of the present invention can be preferably represented by the following formula 3 or formula 4. [
[Formula 3]
5'-P Xa- LQ H -3 '
[Formula 4]
5'-Q H -LP Xa -3 '
In the above formula 3 and formula 4, P Xa is an RNA aptamer for blood coagulation factor Xa, and preferably 6, 7, 8, 9, 12, 22, 27, 28, 29, 30 out of the continuous nucleotide sequence of SEQ ID NO: , 32 and 34 bases were modified to 2'-O-methylcytidine or 2'-fluorocytidine and 17, 20, 23, 24, 33, The 35th and 36th bases consist of a contiguous base sequence modified with 2'-O-methyluridine or 2'-fluorouridine. Further, the above-mentioned Q H is a DNA aptamer for hemin, and preferably consists of the sequential base sequence of SEQ ID NO: 4. The L is a linker molecule in the form of an oligonucleotide and preferably consists of a contiguous nucleotide sequence of SEQ ID NO: 5, a contiguous nucleotide sequence of SEQ ID NO: 6, a contiguous nucleotide sequence of SEQ ID NO: 7 or a contiguous nucleotide sequence of SEQ ID NO: .
The novel double aptamer for the detection of blood coagulation factor Xa represented by the above formula 3 is more preferably selected from the group consisting of 6, 7, 8, 9, 12, 22, 27, 28, 29, 30, The 32nd and 34th bases were modified to 2'-O-methylcytidine or 2'-fluorocytidine and 17, 20, 23, 24, 33, 35 And a continuous base sequence in which the 36th base is modified with 2'-O-methyluridine or 2'-fluorouridine. The novel double aptamer for detecting blood coagulation factor Xa represented by the above formula 4 is more preferably selected from the group consisting of 37, 38, 39, 40, 43, 53, 58, 59, 60, 61, 63 and 65 bases were modified with 2'-O-methylcytidine or 2'-fluorocytidine and 48, 51, 54, 55, 64, 66 And a continuous base sequence in which the 67th base is modified with 2'-O-methyluridine or 2'-fluorouridine.
The novel double aptamer for detecting blood coagulation factor Xa according to another example of the present invention competitively binds to blood coagulation factor Xa and hemin and when the aptamer portion for blood coagulation factor Xa binds to blood coagulation factor Xa The aptamer moiety for hemin is less responsive to hemin. In addition, when hemin binds to the aptamer portion of hemin, a DNA-based enzyme in the form of a quadruplex structure is formed. Since the DNA-based enzyme has a peroxidase activity, it can replace horseradish peroxidase and the like. On the other hand, when the blood coagulation factor Xa is present in the assay sample in a relatively larger amount than hemin, the novel double aptamer for detecting coagulation factor Xa according to another embodiment of the present invention exhibits weak peroxidase activity, When the blood coagulation factor Xa is present in the assay sample in a relatively small amount compared to hemin, the novel double aptamer for detecting coagulation factor Xa according to another embodiment of the present invention exhibits a strong peroxidase activity. The blood coagulation factor Xa present in the blood sample can be quantitatively detected using the above-described characteristics of the novel dual appamer for detecting blood coagulation factor Xa according to another example of the present invention.
Another aspect of the present invention relates to a method for quantitatively and reliably detecting a trace amount of blood coagulation factor Xa present in a blood sample. A method for detecting blood coagulation factor Xa according to another embodiment of the present invention comprises the steps of adding a blood sample to a solution containing both double aptamer for detecting coagulation factor Xa and hemin and reacting to form a first reaction mixture ; Adding a chromogenic substrate and hydrogen peroxide to the first reaction mixture and reacting to form a second reaction mixture; And measuring the chromaticity or absorbance of the second reaction mixture. The coloring substrate is not limited in its kind as long as it can be converted into a predetermined coloring substance by reacting with hydrogen peroxide in the presence of a DNA-based enzyme having peroxidase or peroxidase activity, For example, TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], luminol, isoluminol ), Amplex-Red (10-acetyl-3,7-dihydroxyphenoxazine) or Amplex-UltraRed, and Amplex-Red (10-acetyl-3,7-dihydroxyphenoxazine) or Amplex-UltraRed. Amplex-Red (10-acetyl-3,7-dihydroxyphenoxazine) or Amplex-UltraRed can be used in a 1: 1 stoichiometric ratio with hydrogen peroxide in the presence of DNA-based enzymes (peroxidase or peroxidase) To generate a red fluorescent material, resorufin.
According to another preferred embodiment of the present invention, a method for detecting blood coagulation factor Xa comprises the steps of: adding a blood sample to a solution containing both of a dual appamer for detecting coagulation factor Xa and hemin, ; Adding a chromogenic substrate and hydrogen peroxide to the first reaction mixture and reacting to form a second reaction mixture; Adding a luminous inducer and hydrogen peroxide to the second reaction mixture and reacting to form a third reaction mixture; And measuring the emission intensity of the third reaction mixture. According to another preferred embodiment of the present invention, in the method for detecting blood coagulation factor Xa, the chromogenic substrate reacts with hydrogen peroxide in the presence of a DNA-based enzyme exhibiting peroxidase activity or peroxidase activity, For example, TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline 6-sulfonic acid], luminol, isoluminol, 10-acetyl-3,7-dihydroxyphenoxazine or Amplex-UltraRed, and Amplex-Red (10-acetyl -3,7-dihydroxyphenoxazine) or Amplex-UltraRed. Amplex-Red (10-acetyl-3,7-dihydroxyphenoxazine) or Amplex-UltraRed can be used in a 1: 1 stoichiometric ratio with hydrogen peroxide in the presence of DNA-based enzymes (peroxidase or peroxidase) To generate a red fluorescent material, resorufin. In addition, the luminescent inducing material reacts with hydrogen peroxide to generate a high-energy intermediate material, and through the high-energy intermediate material, energy is transferred to a chromogenic fluorescent material, which is a product of the chromogenic substrate. Ultimately, The type of the chemiluminescence is not limited as long as it releases chemiluminescence. For example, ODI (1,1'-oxalyldiimidazole), OD2MI (1,1'-oxalyldi-2-methyl- imidazole) 1-oxalyldi-4-methyl-imidazole), OD2EI (1,1'-oxalyldi-2-ethyl- imidazole), OD4EI (1,1'- 1'-oxalyldisodium benzoate, OD2B (1,1'-oxalyldi-2-sodium benzoate) or OD4B (1,1'-oxalyldi-4-sodium benzoate). The present invention refers to both the disclosures of U.S. Patent Application Publication No. 2004/0142358 and U.S. Patent No. 8,492,101 concerning chemiluminescence methods using ODI, ODI derivatives, ODB or ODB derivatives. FIG. 3 schematically shows a method for detecting blood coagulation factor Xa according to another preferred embodiment of the present invention. As shown in FIG. 3, when a double aptamer and hemin for detecting blood coagulation factor Xa are injected into blood or plasma in which blood coagulation factor Xa is present, blood coagulation factor Xa and hemin competitively bind blood coagulation factor Xa And reacts with a double-app tamer for detection. When the concentration of blood coagulation factor Xa increases, the concentration of DNAzyme for binding to hemin decreases and the intensity of light weakens. Therefore, the intensity of the chemiluminescence that emits red light is inversely proportional to the concentration of blood coagulation factor Xa present in the blood or plasma.
Another aspect of the present invention relates to a kit capable of quantitatively and reliably detecting a trace amount of blood coagulation factor Xa present in a blood sample. A kit for detecting blood coagulation factor Xa according to another embodiment of the present invention includes a solution containing a double aptamer for detecting blood coagulation factor Xa, a solution containing hemin, a solution containing chromogenic substrate and a solution containing hydrogen peroxide. The method for detecting blood coagulation factor Xa according to another embodiment of the present invention can be implemented using a kit for detecting blood coagulation factor Xa according to another example of the present invention. Further, a kit for detecting blood coagulation factor Xa according to another preferred embodiment of the present invention is a kit for detecting blood coagulation factor Xa, comprising a solution containing a double aptamer for detecting blood coagulation factor Xa, a solution containing hemin, a solution containing a chromogenic substrate, And hydrogen peroxide-containing solution. The method for detecting blood coagulation factor Xa according to another preferred embodiment of the present invention can be implemented using a kit for detecting blood coagulation factor Xa according to another preferred embodiment of the present invention. In the kit for the detection of blood coagulation factor Xa of the present invention, the chromogenic substrate is preferably TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline- 6-sulfonic acid], luminol, isoluminol, Amplex-Red (10-acetyl-3,7-dihydroxyphenoxazine) or Amplex-UltraRed. The light emitting inducing material is preferably selected from the group consisting of 1,1'-oxalyldiimidazole (ODI), 1,1'-oxalyldi-2-methyl-imidazole (OD2MI), and 1,1'-oxalyldi- ), OD2EI (1,1'-oxalyldi-2-ethyl-imidazole), OD4EI (1,1'-oxalyldi-4-ethyl- imidazole), ODB (1,1'-oxalyldisodium benzoate) '-oxalyldi-2-sodium benzoate) or OD4B (1,1'-oxalyldi-4-sodium benzoate).
Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.
Ⅰ. Double for detection of coagulation factor Ⅱa Aptamer And detection of blood clotting factor Ⅱa
1. Preparation of double-app tamer for blood coagulation factor IIa detection
A dual appamer for the detection of blood coagulation factor IIa described in Table 1 was prepared using SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process.
The double aptamer of Table 1 is a DNA for a hematin (hemin) consisting of 5'-agagggtagggtaaat-3 'and a DNA aptamer for blood coagulation factor IIa consisting of 5'-ggttggtgtggttgg-3' or 5'- agtccgtggtagggcaggttggggtgac- Aptamer has a structure bound by an oligonucleotide linker molecule composed of 5'-ttttt-3 ', 5'-taaat-3' or 5'-taaaat-3 '.
2. Detection of blood clotting factor Ⅱa using double-app tamer
(1) Test Example 1
1) An aqueous solution of a double aptamer (selected from dual aptamer No. 1 to double aptamer No. 4) at a concentration of 1 μM and an aqueous solution of hemin at a concentration of 285 ng / ml were mixed at a volume ratio of 1: 1 to obtain 0.1 ml Of a double aptamer-hemin mixed solution.
2) To the double-aptamer-hemin mixed solution, 0.05 ml of a 200 M concentration blood coagulation factor Ⅱa aqueous solution or 0.05 ml of distilled water not containing the coagulation factor Ⅱa was added and reacted at room temperature for 30 minutes to form a first reaction mixture Respectively.
3) 0.15 ml of a solution containing Amplex-Red at a concentration of 1 μM and hydrogen peroxide at a concentration of 0.4 mM was added to the first reaction mixture and reacted for 5 minutes to form a second reaction mixture. A predetermined amount of resorufin was produced in the second reaction mixture.
4) In the presence of 0.01 mM of TCPO [Bis (2,4,6-trichloro) phenyl oxalate] and 0.04 mM of 4M imH (4-Methylimidazole) in an ethyl acetate solvent, OD4MI (1,1'-oxalyldi- imidazole was prepared. Hydrogen peroxide was dissolved in isopropyl alcohol at a concentration of 0.01 M to prepare a solution containing hydrogen peroxide. A hydrogen peroxide-containing solution and a solution containing OD4MI (1,1'-oxalyldi-4-methyl-imidazole) were sequentially added to the second reaction mixture and reacted to prepare a third reaction mixture. In the third reaction mixture, resorufin releases red light by ODI chemiluminescence.
5) The luminescence intensity emitted from the third reaction mixture was measured using a chemiluminescence analyzer (Model: LB 9507; EG & G. Berthold).
Table 2 shows the results of the double-aptamer No. 1 to double appamer No. 1 in the method of Test Example 1. 4 was measured for the coagulation factor Ⅱa.
* CL 0 : chemiluminescence intensity when using samples without blood coagulation factor IIa
* CL F Ⅱa : the intensity of chemiluminescence when 200 μM blood clotting factor Ⅱa-containing samples were used
As shown in Table 1, the reactivity between the blood coagulation factor IIa and the dual appamer was influenced not only by the aptamer structure for blood coagulation factor IIa but also by the form of the linker. The value of CL F Ⅱa / CL 0 is the double app tamer number. 3 was the smallest. This is a dual app tamer no. 3 reacts most rapidly with blood coagulation factor IIa. Specifically, when blood coagulation factor IIa is present, When the hemoglobin concentration Ⅱa was not present, the intensity of the chemiluminescence measured after the reaction of 3 and hemin did not change. 3 and heme were about 80% smaller than those measured after chemiluminescence. These results show that the dual app tamer no. 3 shows that a low concentration of coagulation factor IIa can be quantified. In addition, the reactivity of the double aptamer to blood coagulation factor IIa was improved when the adenine (A) base was used as a linker component rather than the thymine (T) base. Specifically, a dual appamer No. 1 having a linker consisting of an adenine (A) base and a thymine (T) base. A double-app tamer No. 1 having a linker consisting of only four Thymine (T) bases. CL FIIa / CL 0 values smaller than 2.
On the other hand, 5 is the double app tamer No. 3, the position of the aptamer for the aptamer and the hematin relative to the coagulation factor Ⅱa was changed through the linker. 6 is a double app tamer number. 4, the position of the aptamer for the appetamer and hemin relative to the coagulation factor Ⅱa was changed only through the linker. Therefore, 5 and dual app tamer no. 6 can also be used to determine blood levels of coagulation factor IIa.
(2) Test Example 2
1) Double App Tarmer No. 3 was dissolved in PBS buffer to prepare a 200 nM double aptamer solution.
2) An aqueous solution of blood clotting factor IIa with various concentrations from 0 nM to 400 nM was prepared.
3) 0.05 ml of double-aptamer solution and 0.05 ml of blood coagulation factor IIa solution were mixed and reacted for 30 minutes to form a first reaction mixture.
4) To the first reaction mixture was added 0.05 ml of an aqueous solution of hemin (285 ng / ml) and reacted for 5 minutes to form a second reaction mixture.
5) To the second reaction mixture, 0.15 ml of a solution containing Amplex-Red at a concentration of 1 μM and hydrogen peroxide at a concentration of 0.4 mM was added and reacted for 5 minutes to form a third reaction mixture. A predetermined amount of resorufin was produced in the third reaction mixture.
6) In the presence of 0.01 mM of TCPO [Bis (2,4,6-trichloro) phenyl oxalate] and 0.04 mM of 4MImH (4-Methylimidazole) in ethyl acetate, OD4MI (1,1'-oxalyldi- imidazole was prepared. Hydrogen peroxide was dissolved in isopropyl alcohol at a concentration of 0.01 M to prepare a solution containing hydrogen peroxide. A hydrogen peroxide-containing solution and a solution containing OD4MI (1,1'-oxalyldi-4-methyl-imidazole) were sequentially added to the third reaction mixture and reacted to prepare a fourth reaction mixture. In the fourth reaction mixture, resorufin emits red light by ODI chemiluminescence.
7) The luminescence intensity emitted from the fourth reaction mixture was measured using a chemiluminescence analyzer (Model: LB 9507; EG & G. Berthold).
FIG. 4 is a graph showing the relationship between the concentration of blood coagulation factor IIa and the intensity of chemiluminescence, which are present in the assay sample, on the basis of the result of the measurement of the reactivity of the double aptamer No. 3 to blood coagulation factor IIa by the method of Test Example 2 It is a graph. 5 is a graph showing the relationship between the concentration of blood coagulation factor IIa present in the assay sample and the level of chemiluminescence intensity It is a graph plotted with reciprocal number. As shown in FIG. 4, the intensity of chemiluminescence decreased as the concentration of blood coagulation factor IIa in the assay sample increased. As shown in FIG. 5, when the concentration of the blood coagulation factor IIa and the inverse of the chemiluminescence intensity were plotted, a calibration curve of the first-order equation was obtained. Therefore, when using the dual aptamer No. 3 or the like, the concentration of blood coagulation factor IIa present in blood or plasma can be determined quickly and accurately.
Ⅱ. Double for detection of blood coagulation factor Xa Aptamer And detection of blood coagulation factor Xa using the same
1. Preparation of dual appamer for detection of blood coagulation factor Xa
A dual-app tamer for the detection of coagulation factor Xa described in Table 3 below was prepared using SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process. In Table 3 below, c m represents 2'-O-methylcytidine and u m represents 2'-O-methyluridine.
Dual aepta of Table 1 dimmer gagagc m c m c m-5'c agc m m m gagau aau ac m m u m u m ggc m c m c m c m m c gc m u m u m u m -3 And a DNA aptamer for hemin consisting of 5'-aaagggtagggcgggttgggtaaat-3 'linked by an oligonucleotide linker molecule consisting of 5'-aaaaat-3' I have.
2. Detection of blood clotting factor Xa using double-app tamer
(1) Test Example 3
1) An aqueous solution of double aptamer No. 7 at a concentration of 200 nM and an aqueous solution of hemin at a concentration of 285 ng / ml were mixed at a volume ratio of 1: 1 to prepare 0.1 ml of a double aptamer-hemin mixed solution.
2) 0.05 ml of aqueous solution of blood coagulation factor Xa at a concentration of 100 nM or 0.05 ml of distilled water containing no blood coagulation factor Xa was added to the double-aptamer-hemin mixed solution and reacted at room temperature for 30 minutes to form a first reaction mixture Respectively.
3) 0.15 ml of a solution containing Amplex-Red at a concentration of 1 μM and hydrogen peroxide at a concentration of 0.4 mM was added to the first reaction mixture and reacted for 5 minutes to form a second reaction mixture. A predetermined amount of resorufin was produced in the second reaction mixture.
4) In the presence of 0.01 mM of TCPO [Bis (2,4,6-trichloro) phenyl oxalate] and 0.04 mM of 4M imH (4-Methylimidazole) in an ethyl acetate solvent, OD4MI (1,1'-oxalyldi- imidazole was prepared. Hydrogen peroxide was dissolved in isopropyl alcohol at a concentration of 0.01 M to prepare a solution containing hydrogen peroxide. A hydrogen peroxide-containing solution and a solution containing OD4MI (1,1'-oxalyldi-4-methyl-imidazole) were sequentially added to the second reaction mixture and reacted to prepare a third reaction mixture. In the third reaction mixture, resorufin releases red light by ODI chemiluminescence.
5) The luminescence intensity emitted from the third reaction mixture was measured using a chemiluminescence analyzer (Model: LB 9507; EG & G. Berthold).
Fig. 6 is a graph showing the results obtained by the method of Test Example 3; 7 < / RTI > for blood coagulation factor Xa. In Fig. 6, the left side shows the result of the analytical sample containing no blood coagulation factor Xa and the right side shows the result of the analytical sample containing the blood coagulation factor Xa. As shown in FIG. 6, a large amount of DNAzyme was produced in the analytical sample not containing the coagulation factor Xa, and the colorless Amplex Red was transformed into a pink resorufin. However, in analytical samples containing blood coagulation factor Xa, a small amount of DNAzyme was produced and little pink resorufin was observed.
(2) Test Example 4
1) Double App Tarmer No. 7 was dissolved in PBS buffer to prepare a 200 nM double aptamer solution.
2) An aqueous solution of blood clotting factor Xa having various concentrations from 0 nM to 400 nM was prepared.
3) 0.05 ml of the double-aptamer solution and 0.05 ml of the blood coagulation factor Xa aqueous solution were mixed and reacted for 30 minutes to form the first reaction mixture.
4) To the first reaction mixture was added 0.05 ml of an aqueous solution of hemin (285 ng / ml) and reacted for 5 minutes to form a second reaction mixture.
5) To the second reaction mixture, 0.15 ml of a solution containing Amplex-Red at a concentration of 1 μM and hydrogen peroxide at a concentration of 0.4 mM was added and reacted for 5 minutes to form a third reaction mixture. A predetermined amount of resorufin was produced in the third reaction mixture.
6) In the presence of 0.01 mM of TCPO [Bis (2,4,6-trichloro) phenyl oxalate] and 0.04 mM of 4MImH (4-Methylimidazole) in ethyl acetate, OD4MI (1,1'-oxalyldi- imidazole was prepared. Hydrogen peroxide was dissolved in isopropyl alcohol at a concentration of 0.01 M to prepare a solution containing hydrogen peroxide. A hydrogen peroxide-containing solution and a solution containing OD4MI (1,1'-oxalyldi-4-methyl-imidazole) were sequentially added to the third reaction mixture and reacted to prepare a fourth reaction mixture. In the fourth reaction mixture, resorufin emits red light by ODI chemiluminescence.
7) The luminescence intensity emitted from the fourth reaction mixture was measured using a chemiluminescence analyzer (Model: LB 9507; EG & G. Berthold).
FIG. 7 is a graph showing the relationship between the concentration of blood coagulation factor Xa present in the assay sample and the chemiluminescence intensity of the assay sample, based on the result of measuring the reactivity of the double-aptamer No. 7 to blood coagulation factor Xa by the method of Test Example 4. FIG. It is a graph. 8 is a graph showing the relationship between the concentration of blood coagulation factor Xa present in the assay sample and the level of chemiluminescence intensity in the assay sample after measuring the reactivity of the double-aptamer No. 7 to blood coagulation factor Xa by the method of Test Example 4. FIG. It is a graph plotted with reciprocal number. As shown in FIG. 7, as the concentration of blood coagulation factor Xa in the assay sample increased, the intensity of chemiluminescence decreased. As shown in FIG. 8, when the concentration of blood coagulation factor Xa and the reciprocal of the chemiluminescence intensity were plotted, a calibration curve of the first-order equation was obtained. Therefore, when the double abdominal No. 7 or the like is used, the concentration of blood coagulation factor Xa present in blood or plasma can be determined quickly and accurately.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Therefore, the scope of the present invention should be construed as including all embodiments falling within the scope of the appended claims.
<110> MIRUSYSTEMS CO., LTD KIM, Chan-il <120> Dual aptamer for detecting Blood coagulation factor IIa and use <130> DP-16-162 <160> 15 <170> KoPatentin 3.0 <210> 1 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer 1 against blood coagulation factor IIa <400> 1 ggttggtgtg gttgg 15 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer 2 against blood coagulation factor IIa <400> 2 agtccgtggt agggcaggtt ggggtgac 28 <210> 3 <211> 36 <212> RNA <213> Artificial Sequence <220> <223> RNA aptamer against blood coagulation factor Xa <400> 3 gagagcccca gcgagauaau acuuggcccc multiplier 36 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer against hemin <400> 4 aaagggtagg gcgggttggg taaat 25 <210> 5 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide linker 1 <400> 5 ttttt 5 <210> 6 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide linker 2 <400> 6 commitment 5 <210> 7 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide linker 3 <400> 7 taaaat 6 <210> 8 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide linker 4 <400> 8 aaaaat 6 <210> 9 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Dual aptamer 1 for detecting blood coagulation factor IIa <400> 9 ggttggtgtg gttggttttt aaagggtagg gcgggttggg taaat 45 <210> 10 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Dual aptamer 2 for detecting blood coagulation factor IIa <400> 10 agtccgtggt agggcaggtt ggggtgactt ttttaaaggg tagggcgggt tgggtaaat 59 <210> 11 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Dual aptamer 3 for detecting blood coagulation factor IIa <400> 11 ggttggtgtg gttggtaaat aaagggtagg gcgggttggg taaat 45 <210> 12 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Dual aptamer 4 for detecting blood coagulation factor IIa <400> 12 agtccgtggt agggcaggtt ggggtgacta aaataaaggg tagggcgggt tgggtaaat 59 <210> 13 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Dual aptamer 5 for detecting blood coagulation factor IIa <400> 13 aaagggtagg gcgggttggg taaattaaat ggttggtgtg gttgg 45 <210> 14 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Dual aptamer 6 for detecting blood coagulation factor IIa <400> 14 aaagggtagg gcgggttggg taaattaaaa tagtccgtgg tagggcaggt tggggtgac 59 <210> 15 <211> 67 <212> DNA_RNA <213> Artificial Sequence <220> <223> Dual aptamer 7 for detecting blood coagulation factor Xa <220> <223> RNA aptamer against blood coagulation factor Xa (seq no.3) -Oligonucleotide linker 4 (seq no. 8) -DNA aptamer against hemin (seq # 4) <400> 15 gagagcccca gcgagauaau acuuggcccc gcucuuaaaa ataaagggta gggcgggttg 60 ggtaaat 67
Claims (16)
[Formula 1]
5'-P IIa- LQ H -3 '
[Formula 2]
5'-Q H -LP IIa -3 '
In the above Equations 1 and 2, P IIa is a DNA aptamer for blood coagulation factor IIa, Q H is a DNA aptamer for hemin and L is a linker molecule in the form of an oligonucleotide.
Adding a chromogenic substrate and hydrogen peroxide to the first reaction mixture and reacting to form a second reaction mixture; And
And measuring the chromaticity or absorbance of the second reaction mixture.
Adding a chromogenic substrate and hydrogen peroxide to the first reaction mixture and reacting to form a second reaction mixture;
Adding a luminous inducer and hydrogen peroxide to the second reaction mixture and reacting to form a third reaction mixture; And
Lt; RTI ID = 0.0 > IIa < / RTI > comprising measuring the luminescence intensity of the third reaction mixture.
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