KR20100093502A - Method for detecting target molecules using aptamers - Google Patents

Abstract

PURPOSE: A method for detecting a target material using an aptamer is provided to detect material of low molecule and to reduce detection cost using the cheap aptamer. CONSTITUTION: A method for detecting a target material using an aptamer comprises: a step of adding a sample containing the target material and a second aptamer in which specific labeling material is attached to a first aptamer which specifically binds to the target material; and a step of analyzing the labeling material to detect the target material. The sample is collected from water, blood, urine, tear, sweat, lymph, cerebrospinal fluid, soil, air, food, waste, tissue of animal and plant. The target material is bisphenol A.

Description

Method for Detecting Target Materials Using Aptamers {Method for Detecting Target Molecules Using Aptamers}

The present invention relates to a method for detecting a target material using an aptamer and a kit for detection. More specifically, a first aptamer and a target material are added by adding a sample and a second aptamer to a first aptamer fixed to a solid phase. And a method for detecting a target material and a kit for detecting the sandwich material between the second aptamer.

Developing new sensor platforms for sensitive and specific detection of small molecules in solution is critical for the monitoring of disease-related metabolites, environmental contaminants and food toxins. Until recently, analysis of low molecular weight analytes such as metabolites, environmental contaminants, and toxins has generally gone through complex experiments, such as GC / MS or HPLC, which takes a long time and even on-site analysis. -site analysis) (Stales, CA et al, Environ. Toxicol. Chem. , 20: 2450, 2001). In order to succeed on-site real-time detection, small scale devices and specifically binding reagents were required.

As a portable platform for detecting small substances, a detection platform such as surface plasmon resonance is known, but it is known that it is difficult to detect an extremely low molecular weight substance having a molecular weight of 400 or less, and moreover, 17 beta 1 μM (272 ppb) concentration analysis using estradiol (β-estradiol, MW: 272) binding specific aptamers, it was reported that it is impossible to analyze the concentration of such a low molecular weight material as shown in FIG. Kim et al., Biosens. Bioelecrtron. , 22: 2525, 2007). Therefore, there has been a demand for the development of a new platform that can specifically and sensitively detect low molecular weight materials.

In addition, specific and sensitive detection of analytes requires reagents that specifically bind to analytes such as antibodies. However, small, low molecular weight substances are typically too small or too toxic to generate antibodies in animals, so that new specific binding reagents targeting small molecules are required.

On the other hand, aptamer is a single-chain DNA or RNA molecule, refers to a small single-stranded oligonucleic acid that can specifically recognize the target material with high affinity. Aptamers have been recognized as alternatives to antibodies because they can be used as a component of biosensors that can recognize molecules in detection and analysis systems. In particular, aptamers can be used as target molecules of various organic and inorganic substances, including toxins, unlike antibodies, and once isolated, the aptamers are reproduced with low cost and consistency by automated oligomer synthesis method. This was possible and economical. Since the development of aptamer-based biosensors that measured target proteins using fluorescently labeled aptamers in 1996, various aptamer biosensors have been developed based on the advantages and structural characteristics of such aptamers. Yeon-seok Kim & Man-bok Koo, NICE, 26 (6): 690, 2008).

However, conventional analytical methods using aptamers have been used in a competitive analytical method such as detaching or combining aptamers attached to a specific material. There has been a demand for the development of a detection method.

Accordingly, the present inventors have diligently tried to provide a new detection method capable of detecting small molecules, especially small molecules present in a solution. As a result, the inventors added a sample and a second aptamer to the first aptamer immobilized on the solid phase, After forming a sandwich bond between the 1 aptamer, the target material and the second aptamer, it was confirmed that a nonpolar substance having a low molecular weight such as bisphenol A could be detected, thereby completing the present invention.

It is an object of the present invention to provide a method for detecting a new target substance which can also detect low molecular weight substances present in a solution.

Another object of the present invention is to provide a kit for detecting a target substance, which can also detect low molecular weight substances present in a solution.

In order to achieve the above object, the present invention provides a method for detecting a target material using an aptamer, comprising the following steps:

(a) a sample containing a target material in a first aptamer fixed in a solid phase and specifically binding to the target material; And reacting by adding a second aptamer specifically bound to the target material and having a label attached thereto. And

(b) detecting the target substance by analyzing the labeling substance.

The present invention also provides a method for detecting a target substance using an aptamer, comprising the following steps:

(a) reacting the first aptamer fixed to the solid phase and specifically binding to the target material, adding a sample containing the target material and a second aptamer specifically binding to the target material;

(b) binding a label to the second aptamer; And

(c) detecting the target substance by analyzing the labeling substance.

The present invention also provides a kit for detecting a target material comprising a detection reagent comprising a solid phase to which a first aptamer specifically binding to a target material is fixed, and a second aptamer specifically binding to the target material. to provide.

The present invention also includes a detection reagent containing a solid phase in which a first aptamer specifically binding to bisphenol A is immobilized, and a second aptamer specifically binding to bisphenol A to which a labeling substance is attached. The first aptamer or the second aptamer provides a kit for detecting bisphenol A, characterized in that selected from aptamers represented by the nucleic acid sequence of SEQ ID NO: 2 to 28.

The present invention also provides a nucleic acid fragment for aptamer label, characterized in that the label is attached and capable of complementarily binding to the aptamer end.

The present invention has the effect of providing a new target material detection method and kit for detection using aptamers. The detection method according to the present invention enables the detection of low molecular weight substances, which have been difficult to detect in the past, to detect metabolites, environmental contaminants, food toxins, and the like, which are associated with diseases present in the solution, and are directly and easily As a method, it is very useful because it can improve the economics by using aptamer which can be reproduced consistently and produced at low cost.

FIG. 1 shows the results of 17 beta-estradiol detection using aptamer according to a conventional method using SPR.
2 is a schematic diagram of a process for binding bisphenol A and labeled aptamer to an immobilized aptamer according to the method according to the invention.
3 is a result of performing bisphenol A detection through sandwich bonding according to the method according to the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

Definitions of main terms used in the detailed description of the present invention are as follows.

As used herein, "aptamer" refers to a small single-stranded oligonucleotide that can specifically recognize a target with high affinity.

“Sample” as used herein is a composition that contains or is believed to contain a target material of interest and is to be analyzed, from samples taken from any one or more of liquid, soil, air, food, waste, flora and fauna and flora and fauna. It may be characterized in that the detection, but is not limited thereto. At this time, the liquid may be characterized in that the water, blood, urine, tears, sweat, saliva, lymph and cerebrospinal fluid, and the water, such as precipitation (江水), sea water (海水), lakes (우수 水) and rain (Sunsu) Including, waste includes sewage, wastewater, and the like, the flora and fauna includes the human body. In addition, the animal and plant tissues include tissues such as mucous membranes, skin, skin, hair, scales, eyes, tongue, cheeks, hoofs, beaks, snouts, feet, hands, mouths, nipples, ears, and nose.

The present invention relates to a method for detecting a target material using an aptamer, which includes, in one aspect, the following steps:

(a) a sample containing a target material in a first aptamer fixed in a solid phase and specifically binding to the target material; And reacting by adding a second aptamer specifically bound to the target material and having a label attached thereto. And

(b) detecting the target substance by analyzing the labeling substance.

 As used herein, the "solid phase" is a solid support in which the aptamer is fixed, and as long as the aptamer can be fixed, its shape or material is not limited. Multi-well type microplates may generally be used for the convenience of performing analytical methods, but other shapes may be used, such as sensor chips, plastics, polypropylene, or columns filled with beads such as Sepharose or Agarose.

The first aptamer may be immobilized by the usual method on the solid phase. In one embodiment of the present invention is fixed by the sol-gel dispensing method, but is not limited to this will be apparent to those of ordinary skill in the art.

In the present invention, the step (a) is a step of reacting the sample and the second aptamer by the addition of the fixed first aptamer, where "reaction" is the first aptamer sample and the second aptamer Addition and incubation means that the target material present in the sample binds to the fixed first aptamer, and the second aptamer reacts to bind to the target material. In this case, the sample and the second aptamer may be mixed first, and then added to the first aptamer. That is, the sample and the second aptamer are mixed to allow specific binding between the target material and the second aptamer in the sample, and then the bound target material-second aptamer is added to the fixed first aptamer. 1 may be combined with the aptamer.

In addition, the step (a) may be characterized in that the sample is first added, and then the second aptamer is added. That is, the target material in the sample may be first bonded to the first aptamer fixed to the solid phase, and then the second aptamer may be sequentially added to bind, thereby allowing the sequential coupling reaction to be performed.

The labeling material attached to the second aptamer may be attached by an indirect or direct method, and a fluorescent material, a radioisotope, etc. may be used as the labeling material, but is not limited thereto. At this time, the type of the fluorescent material is not particularly limited, and for example, a known fluorescent material such as a fluorescent dye such as Cy3 or Cy5, a fluorescent protein such as luciferase or GFP may be used.

In the present invention, the step (b) is a step of detecting the target material by analyzing the labeling material, the change of the labeling material generated when the target material and the second aptamer is bound to the first aptamer fixed to a solid phase Analyze the target material will be detected. At this time, the analysis of the labeling material can be made by a generally known labeling material analysis method. For example, when a fluorescent material is used as a label, emission or color change occurs in the presence of the target material, and thus the target material can be detected by measuring the same. For example, it is possible to check whether a target substance is detected by scanning a well that caused a reaction through an image scanner capable of detecting a fluorescent dye, and the amount of detection can be measured by measuring the degree of thickening of the image through software.

On the other hand, in addition to using the labeling material attached to the second aptamer, in step (a) by combining the sample and the second aptamer to the first aptamer, and then the labeling material by binding to the second aptamer target The present invention relates to a method for detecting a target material using an aptamer, which can detect a substance, and thus, in another aspect, comprising the following steps:

(a) a sample containing a target material in a first aptamer fixed in a solid phase and specifically binding to the target material; And reacting by adding a second aptamer specifically binding to the target material;

(b) binding a label to the second aptamer; And

(c) detecting the target substance by analyzing the labeling substance.

In the present invention, the binding of the labeling material of step (b) may be performed by binding the nucleic acid fragments complementarily to the second aptamer to the second aptamer. .

In this case, "complementary binding" means that the sequence of the synthesized nucleic acid fragment is about 80-90% or more, more preferably about 90-95% or more, even more preferably about 95-99 with respect to the second aptamer % Or more sequences are complementary or completely complementary to each other, said nucleic acid fragment is preferably characterized in that the complementary binding to the end of the second aptamer, wherein the second Aptamers may include additional sequences at the ends for such binding.

In one embodiment of the present invention, in order to check whether the detection method according to the present invention can detect a low molecular weight substance in a solution, it is tested whether the detection of bisphenol A, which is well known as an environmental hormone, is known to be extremely difficult to detect. It was. As a result, it was confirmed that low molecular weight bisphenol A was also specifically detected. This means that the detection method according to the present invention enables the detection of metabolites, toxins, and the like in the solution by using the method according to the present invention, unlike the conventional method of detecting metabolites, toxins, etc. in a solution.

The aptamer used to detect the bisphenol A of the above embodiment may be preferably selected from aptamers represented by nucleic acid sequences of SEQ ID NOs: 2 to 28. In this case, the nucleic acid aptamer is provided as a single-stranded DNA or RNA, when the nucleic acid is RNA, T is represented by U in the nucleic acid sequence, and such a sequence is included in the scope of the present invention. It will be obvious to those with ordinary knowledge in the field.

On the other hand, the method according to the invention may be provided in the form of a kit, in order to increase portability. That is, in another aspect, the present invention includes a detection reagent containing a solid phase in which a first aptamer specifically binding to a target material is immobilized on a solid phase, and a second aptamer specifically binding to the target material. The present invention relates to a kit for detecting a target substance. In this case, the detection reagent containing the second aptamer may be provided in a separate container or in a reaction part to perform sandwich bonding. In addition to the above, the detection kit may include a detection buffer solution, etc., and may further include a tool for mixing the detection reagent and the sample solution to be detected.

The target material detection kit may take the form of a bottle, a tub, a small sachet, an envelope, a tube, an ampoule, and the like, which may be partially or wholly plastic, glass, paper, or foil. , Wax and the like. The container may be equipped with a fully or partially separable stopper, which may initially be part of the container or attached to the container by mechanical, adhesive, or other means. The container may also be equipped with a stopper, which may be accessible to the contents by a needle. The kit may include an external package, which may include instructions for use of the components.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Particularly, in the following examples, the detection effect of bisphenol A was confirmed by using an aptamer specifically binding to bisphenol A, but the method according to the present invention was used by using an aptamer specifically binding to another target material. Applicability to the detection of materials will be apparent to those of ordinary skill in the art.

Example 1 Isolation of Aptamers Specificly Bind to Bisphenol A and Preparation of Sol-Gel Chips Fixed Therewith

1-1: Separation of aptamers that specifically bind to bisphenol A

First, a random ssDNA library having the following sequence was chemically synthesized and separated by PAGE (Genotech Inc., Korea).

5'-GGGCCGTTCGAACACGAGCATG-N ₆₀ -GGACAGTACTCAGGTCATCCTAGG-3 '(SEQ ID NO: 1)

For the ssDNA pool, SELEX screening and amplification were performed 12 times using a bisphenol A-agarose affinity column to select a total of 27 bisphenol A specific aptamers.

# 31 5'-CGGCCCTAGG ATGACCTGAG TACTGTCCCT CACCCCTACT TCCGCCACTG GCCCAACAGC-3 '(SEQ ID NO: 2)

# 23 5'-TGCCTAGGAT GACCTGAGTA CTGTCCAGGC TCCGACCTTG TCCCTGCCGC CACTCTCCCA-3 '(SEQ ID NO: 3)

# 47 5'-GCGGACGGGC TCGGCTCACC TAGGATGACC TGAGTACTGT CCCCGTGGCG CTAATTCGGG-3 '(SEQ ID NO: 4)

# 50 5'-CGGCCCGCCC CTAGGATGAC CTGAGTACTG TCCGCGGGAC GGTATCGCTG AGACAGGTGC-3 '(SEQ ID NO: 5)

# 41 5'-CGGCAGCCCT AGGATGACCT GAGTACTGTC CGCGAAAGAC TCCATGGTAC CCGGTGCTTA-3 '(SEQ ID NO: 6)

# 27 5'-GGGGGCGTCG NCCTAGGATG ACCTGAGTAC TGTCCGCACN CAGGGAGGAT GCATTGAC-3 '(SEQ ID NO: 7)

# 45 5'-GTGTCCCCAC GTCCTAGGAT GACCTGAGTA CTGTCCAATG CCGCTCCTCC CGATGCAGAC-3 '(SEQ ID NO: 8)

# 11 5'-CTCTTCNCTC CAATTCGTAA GATGACCTGA GGTCTGCCCA ACGGTGTTTA GAACCCCTTG-3 '(SEQ ID NO: 9)

# 12-3 5'-CGCAGCGCGC CCCTGAGTAC TGTCCGCCCA ACGGTGTGAC GGCCCTGCGA TCAACGATTG-3 '(SEQ ID NO: 10)

# 12-4 5'-GGGCCGTCCT AGGATGACCT GAGTACTGTC CGCCCAACGG TGTGACGGCC CTGCGATCAA-3 '(SEQ ID NO: 11)

# 22 5'-CCCTCGCCCT GAGTACTGTC CCCCGTCCGT CCGGTGAGGG CCACTATCGC TAACTGATCA-3 '(SEQ ID NO: 12)

# 4 5'-AGGCCGTTGG TGTGGTGGGC CTAGGGCCGG CGGCGCACAG CTGTTATAGA CGTCTCCAGC-3 '(SEQ ID NO: 13)

# 12-5 5'-CCGCCGTTGG TGTGGTGGGC CTAGGGCCGG CGGCGCACAG CTGTTATAGA CGTCTCCAGC-3 '(SEQ ID NO: 14)

# 6 5'-CCGCCGTTGG TGTGGTGGGC CTAGGGCCGG CGGCGCACAG CTGTTATAGA CGCCTCCAGC-3 '(SEQ ID NO: 15)

# 12-7 5'-CCGCCGTTGG TGTGGTGGGC CCAGGGCCGG CGGCGCACAG CTGTTATAGA CGCCTCCAGC-3 '(SEQ ID NO: 16)

# 12-2 5'-TGACGGTGGC GTGGAGGGCG CGTATCAATC GTTGATCGCA GGGCCGTCAT ACCGTTGGAG-3 '(SEQ ID NO: 17)

# 12-9 5'-TGACGGTGGC GTGGAGGGCG CGTATCAATC GTTGATCGCA GGGCCGTCAT ACCGTTGGGGG-3 '(SEQ ID NO: 18)

# 12-6 5'-TGACGGTGGC GTGGAGGGCG CGTATCAATC GTTGATCGCA GGGCCGTCAT ACCGGTCGGG-3 '(SEQ ID NO: 19)

# 2 5'-GCCGACAGGG CATGGGACGC TATCAGCGGT GTCAATCGAA TTCCCGCGGC CGCCATGCGG-3 '(SEQ ID NO: 20)

# 14 5'-GGTCCCCGCA GCTCATACGG CGCTCCAGCG TAATCGAATT CCCGCGGCCG CCATGCGGCC-3 '(SEQ ID NO: 21)

# 46 5'-GCGAGTGGCC CATCAGCAGA GCGTAATCCC CACGCACATC GAGTGCCCCC GGCCGGTGCT-3 '(SEQ ID NO: 22)

# 12 5'-GTATTGTCAT TCATATCCTC GTGCTTGCTG TCCTCACCCC ACCCACCAGA ATGGAAA-3 '(SEQ ID NO: 23)

# 13 5'-CCTGGTATTG TCTTGCCAAT CCTCGCCCTG GCTGTCTTAC CCCTCCCCAC CCGCCTGAAG-3 '(SEQ ID NO: 24)

# 48 5'-GTCGACTCGC GGGTACCGTG CTCAATGTCC CAATCCGGGG AAGCGTTTAG ACCCGCAGCC CAC-3 '(SEQ ID NO: 25)

# 40 5'-GTCGCCACTG CGGGTACCGT GCTTGGGCNA CCGATGNACC NTGNNACCGT GTTTNGCC-3 '(SEQ ID NO: 26)

# 3 5'-CCGGTGGGTG GTCAGGTGGG ATAGCGTTCC GCGTATGGCC CAGCGCATCA CGGGTTCGCA CCA-3 '(SEQ ID NO: 27)

# 32 5'-GGGCGGTGGG TGGCGAGTTG TGAGACGCTG GAGGAGGTTG CTGCCCCCGG CACATTGGGA-3 '(SEQ ID NO: 28)

1-2: Preparation of Sol-Gel Chips

A sensor chip in which a first aptamer was fixed was manufactured as follows. That is, the sol-gel spots were negative control, aptamers (aptamers # 3 and # 12-5), positive control, in the order as shown in Figure 3, positive control (P), # 12-5 aptamer Sol-gel chips were prepared by immobilization on PMMA coated 96-wells using OmniGrid Accent Microarrayer (DIGI LAB, USA) in order of # 3 aptamer and negative control (N).

Sol compositions for the sol-gel spots used above were prepared as follows. First, a sol composition was prepared by mixing tetramethyl orthosilicate (TMOS) and methyltrimethoxysilicate (MTMS). Then, 100 mM HCl was prepared as solution I.

Meanwhile, 100 μm SP buffer solution (pH 5.8) and 20 μl of double distilled water (DDW) were mixed, and then the Cy3 antibody (Santa Cruz, USA) for the positive control and PBS buffer for the negative control were mixed with the mixture. Solution II was vortexed and spin-down for 5 seconds by adding 10 µl of the selected aptamers (aptamers # 3 and # 12-5) for the experimental group to prepare solution II.

Then, the sol composition, solution I and solution II were sequentially dispensed and the sol-gel spot was gelled for 13-15 hours to prepare a sol-gel chip.

Example 2: Detection of Bisphenol A Using Sol-Gel Chips

First, aptamers # 3 and # 12-5 were labeled using terminal deoxynucleotidyl transferase (Terminal Deoxynucleotidyl Transferase; Fermentas, Canada) and Cy3-dUTP (GeneChem, Korea) and used in this experiment. However, labeling may be performed by binding fragments such as 5'-GGGCCGTTCGAACACGAGCATG-3 '(SEQ ID NO: 29) to the ends of aptamers # 3 and # 12-5, respectively, used as the second aptamer. By attaching Cy3 to a nucleic acid fragment such as 5′-CATGCTCGTGTTCGAACGGCCC-3 ′ (SEQ ID NO: 30), thereby allowing the nucleic acid fragment labeled Cy3 to the terminal fragment of the second aptamer to bind complementarily. It may be.

Then, 50 μM bisphenol A and 2 μM cy3-labeled aptamer (second aptamer) were added to each well of the sol-gel chip prepared in Example 1-2. That is, as shown in Figure 3 in each well, bisphenol A (BPA) and aptamer # 12-5 in the upper right well, buffer and aptamer # 3 in the lower left well, BPA and pressure in the lower right well Tamer # 3 was treated and incubated. Then, the washing process was observed with a Multi-Image Analyzer (FUJIFILM, Japan).

As a result, as shown in FIG. 3, a signal appears at a position where # 12-5 aptamer and # 3 aptamer is fixed only when bisphenol A is present (indicated by a red circle), and the sol-gel chip according to the present invention. It was confirmed that the bisphenol A can be specifically detected by using. That is, the nonpolar low molecular weight bisphenol A, which has been difficult to detect in the past, was also specifically detected by the method according to the present invention.

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Dongguk University Industry-Academic Cooperation Foundation <120> Method for Detecting Target Molecules Using Aptamers <130> P10-B051 <160> 30 <170> KopatentIn 1.71 <210> 1 <211> 106 <212> DNA <213> Artificial Sequence <220> <223> random ssDNA library <220> <221> misc_feature (222) (13) .. (72) <223> A or G or C or T <400> 1 gggccgttcg aacacgagca tgnnnnnnnn nnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnnnnnnn nnggacagta ctcaggtcat cctagg 106 <210> 2 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 2 cggccctagg atgacctgag tactgtccct cacccctact tccgccactg gcccaacagc 60                                                                           60 <210> 3 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 3 tgcctaggat gacctgagta ctgtccaggc tccgaccttg tccctgccgc cactctccca 60                                                                           60 <210> 4 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 4 gcggacgggc tcggctcacc taggatgacc tgagtactgt ccccgtggcg ctaattcggg 60                                                                           60 <210> 5 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 5 cggcccgccc ctaggatgac ctgagtactg tccgcgggac ggtatcgctg agacaggtgc 60                                                                           60 <210> 6 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 6 cggcagccct aggatgacct gagtactgtc cgcgaaagac tccatggtac ccggtgctta 60                                                                           60 <210> 7 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 7 gggggcgtcg ncctaggatg acctgagtac tgtccgcacn cagggaggat gcattgac 58 <210> 8 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 8 gtgtccccac gtcctaggat gacctgagta ctgtccaatg ccgctcctcc cgatgcagac 60                                                                           60 <210> 9 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 9 ctcttcnctc caattcgtaa gatgacctga ggtctgccca acggtgttta gaaccccttg 60                                                                           60 <210> 10 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 10 cgcagcgcgc ccctgagtac tgtccgccca acggtgtgac ggccctgcga tcaacgattg 60                                                                           60 <210> 11 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 11 gggccgtcct aggatgacct gagtactgtc cgcccaacgg tgtgacggcc ctgcgatcaa 60                                                                           60 <210> 12 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 12 ccctcgccct gagtactgtc ccccgtccgt ccggtgaggg ccactatcgc taactgatca 60                                                                           60 <210> 13 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 13 aggccgttgg tgtggtgggc ctagggccgg cggcgcacag ctgttataga cgtctccagc 60                                                                           60 <210> 14 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 14 ccgccgttgg tgtggtgggc ctagggccgg cggcgcacag ctgttataga cgtctccagc 60                                                                           60 <210> 15 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 15 ccgccgttgg tgtggtgggc ctagggccgg cggcgcacag ctgttataga cgcctccagc 60                                                                           60 <210> 16 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 16 ccgccgttgg tgtggtgggc ccagggccgg cggcgcacag ctgttataga cgcctccagc 60                                                                           60 <210> 17 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 17 tgacggtggc gtggagggcg cgtatcaatc gttgatcgca gggccgtcat accgttggag 60                                                                           60 <210> 18 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 18 tgacggtggc gtggagggcg cgtatcaatc gttgatcgca gggccgtcat accgttgggg 60 g 61 <210> 19 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 19 tgacggtggc gtggagggcg cgtatcaatc gttgatcgca gggccgtcat accggtcggg 60                                                                           60 <210> 20 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 20 gccgacaggg catgggacgc tatcagcggt gtcaatcgaa ttcccgcggc cgccatgcgg 60                                                                           60 <210> 21 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 21 ggtccccgca gctcatacgg cgctccagcg taatcgaatt cccgcggccg ccatgcggcc 60                                                                           60 <210> 22 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 22 gcgagtggcc catcagcaga gcgtaatccc cacgcacatc gagtgccccc ggccggtgct 60                                                                           60 <210> 23 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 23 gtattgtcat tcatatcctc gtgcttgctg tcctcacccc acccaccaga atggaaa 57 <210> 24 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 24 cctggtattg tcttgccaat cctcgccctg gctgtcttac ccctccccac ccgcctgaag 60                                                                           60 <210> 25 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 25 gtcgactcgc gggtaccgtg ctcaatgtcc caatccgggg aagcgtttag acccgcagcc 60 cac 63 <210> 26 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 26 gtcgccactg cgggtaccgt gcttgggcna ccgatgnacc ntgnnaccgt gtttngcc 58 <210> 27 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 27 ccggtgggtg gtcaggtggg atagcgttcc gcgtatggcc cagcgcatca cgggttcgca 60 cca 63 <210> 28 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> bisphenol A aptamer <400> 28 gggcggtggg tggcgagttg tgagacgctg gaggaggttg ctgcccccgg cacattggga 60                                                                           60 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> fragment attached to 2nd aptamer <400> 29 gggccgttcg aacacgagca tg 22 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Cy3-attached fragment <400> 30 catgctcgtg ttcgaacggc cc 22

Claims (18)

A method for detecting a target material using an aptamer, comprising the following steps:
(a) a sample containing a target material in a first aptamer fixed in a solid phase and specifically binding to the target material; And reacting by adding a second aptamer specifically bound to the target material and having a label attached thereto. And
(b) detecting the target substance by analyzing the labeling substance.
The method of claim 1, wherein the labeling material is a fluorescent material.
The method of claim 2, wherein the analyzing of the fluorescent material is performed by measuring emission or color change of the fluorescent material.
The method of claim 1, wherein the sample is taken from any one or more of water, blood, urine, tears, sweat, saliva, lymph, cerebrospinal fluid, soil, air, food, waste, flora and fauna and flora and fauna. .
The method of claim 1, wherein the first aptamer or the second aptamer is selected from aptamers represented by nucleic acid sequences represented by SEQ ID NOs: 2 to 28. 7.
The method of claim 1 wherein the target material is bisphenol A.
A method for detecting a target material using an aptamer, comprising the following steps:
(a) a sample containing a target material in a first aptamer fixed in a solid phase and specifically binding to the target material; And reacting by adding a second aptamer specifically binding to the target material;
(b) binding a label to the second aptamer; And
(c) detecting the target substance by analyzing the labeling substance.
8. The method of claim 7, wherein step (b) is performed by complementarily binding the nucleic acid fragment to which the label is attached and complementarily binds to the second aptamer. .
The method of claim 8, wherein the nucleic acid fragment binds complementarily with the ends of the second aptamer.
8. The method of claim 7, wherein the labeling substance is a fluorescent substance.
The method of claim 10, wherein the analyzing of the fluorescent material is performed by measuring emission or color change of the fluorescent material.
The method of claim 7, wherein the first aptamer or the second aptamer is selected from aptamers represented by nucleic acid sequences represented by SEQ ID NOs: 2 to 28. 9.
8. The method of claim 7, wherein the target material is bisphenol A.
A target material detection kit comprising a detection reagent containing a solid phase in which a first aptamer specifically binding to a target material is fixed, and a second aptamer specifically binding to the target material.
The kit of claim 14, wherein the second aptamer is combined with a label.
The kit for detecting a target substance according to claim 14, further comprising a nucleic acid fragment having a label attached thereto and complementarily binding to the second aptamer.
A detection reagent comprising a solid phase in which a first aptamer specifically binding to bisphenol A is immobilized, and a second aptamer specifically binding to bisphenol A having a labeling substance attached thereto, wherein the first aptamer Or the second aptamer is selected from aptamers represented by the nucleic acid sequences of SEQ ID NOs: 2 to 28.
An aptamer-labeled nucleic acid fragment, characterized in that the label is attached and capable of complementarily binding to the aptamer end.

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