KR20170109176A - Nucleotide Aptamer for Use in Detection of Bisphenol A - Google Patents

Abstract

The present invention relates to a nucleic acid plasmid for the detection of bisphenol A which is an endocrine disruptor. The nucleic acid aptamer of the present invention is structurally stable, has a long half-life, is easy to synthesize artificially, and can be easily chemically modified at each base sequence position, so that target substances can be detected using various methods. The bisphenol A can be easily detected with high sensitivity when the nucleic acid plasmid, composition, detection kit or detection method for detecting bisphenol A of the present invention is used.

Description

Nucleotide Aptamer for Use in Detection of Bisphenol A

The present invention relates to a nucleic acid plasmid for the detection of bisphenol A which is an endocrine disruptor.

Recently, many kinds of plastic products have been developed and used, and bisphenol A-based polymer materials having suitable physical properties as containers have been widely used. Bisphenol A has been used for more than 50 years for polycarbonate plastics and food cans, bottle caps, food packaging materials, dental resins and other plastic materials used as materials for compact discs, automotive parts, infant feeding bottles, plastic bowls, And has been used as a basic raw material for the synthesis of epoxy resin which is mainly used for the epoxy resin. It is also used as an antioxidant and a vinyl chloride stabilizer in the production of synthetic resins. Because it is used for various purposes, about 20-3 billion kilograms are produced worldwide per year. Approximately 70% of industrially synthesized bisphenol A is used in the synthesis of polycarbonate and about 25% is used in the synthesis of epoxy resins. The remaining 5% is used in phenolic resins, phenolic resins, unsaturated polyester resins, thermal paper antioxidants, polyols, modified polyamides, automotive tires, and flame retardants.

However, in the case of bisphenol A, there is a problem that it is easily liberated. Bisphenol A is liberated from polycarbonate and epoxy resin, and when it is absorbed into human body, it can act like estrogen hormone. Bisphenol A plays a role in disturbing the endocrine system of our body, and even in the presence of trace amounts, bioaccumulation can cause severe disorders such as poor reproductive function, growth disorder, malformation or cancer. In animal experiments, bisphenol A was shown to be similar to estrogen hormones such as 17β-estradiol and ethinylestradiol in vivo, beginning with reports of experimental results in which female estrus was maintained for 40 days when bisphenol A was administered to rats in the 1930s . In vivo experiments, mainly in rats and mice, the uterine weight of immature or ovariectomized experimental animals increased. In addition, animal studies have shown that bisphenol A plays a role similar to estrogen hormone.

Typical human exposure occurs through ingestion of food in contact with packaging materials containing bisphenol A, and in the case of infants, exposure to the hand-mouth route after touching a product containing bisphenol A may occur.

 As a conventional method for detecting bisphenol A, there is a method of detecting the component content by detecting fluorescence signals emitted from bisphenol A using high performance liquid chromatography. This method has the advantage of being highly sensitive and capable of specifically discriminating only one substance, but it is disadvantageous in that it can be detected only by a specific institution equipped with specialized equipment.

Therefore, it is necessary to develop a probe to detect it quickly and easily.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop a nucleic acid plamer probe capable of detecting bisphenol A as an endocrine disruptor. As a result, the present inventors have completed the present invention by confirming that a nucleic acid aptamer containing a predetermined sequence has a binding force to bisphenol A.

Accordingly, an object of the present invention is to provide a nucleic acid plasmid for detecting bisphenol A.

It is still another object of the present invention to provide a composition for detecting bisphenol A which comprises the above-described nucleic acid platemater.

It is still another object of the present invention to provide a bisphenol A detection kit comprising the above-described nucleic acid ablator.

It is still another object of the present invention to provide a bisphenol A detection method using the composition for detecting bisphenol A described above.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a nucleic acid plasmid for detecting bisphenol A comprising the sequence of SEQ ID NO: 9.

The present inventors have made extensive efforts to develop a nucleic acid plamer probe capable of detecting bisphenol A as an endocrine disruptor. As a result, it was confirmed that the nucleotide < RTI ID = 0.0 > aptamer < / RTI >

The bisphenol A (CAS No.: 80-05-7, 4,4'-isopropylidenediphenol, BPA) of the present invention is an organic compound produced by condensation of one molecule of acetone and two molecules of phenol, Such as hydrochloric acid (HCl) or sulfonated polystyrene resin, are used, and a large amount of phenol is used to ensure complete reaction. In the case of bisphenol A of the present invention, there is a problem that it is easily liberated. The liberated bisphenol A acts to disturb the endocrine system of the body, and even in the presence of trace amounts, Cancer, and the like. To pre-detect bisphenol A in order to prevent this problem, the present inventors have developed a nucleic acid plamer probe of a predetermined sequence having a binding force to bisphenol A.

The term "nucleic acid plasmid" of the present invention means a nucleic acid molecule capable of binding a specific molecule with high affinity and specificity, and the nucleic acid means DNA, RNA or nucleic acid variant. The nucleic acid amplimer of the present invention is provided in the form of single stranded DNA, RNA, etc., and the sequence listing No. 9 of the present invention is a DNA stranded DNA sequence, and the nucleic acid sequence of the DNA stranded DNA is replaced with uracil It will be apparent to those skilled in the art that the platelet sequence is included in the scope of the present invention.

The term "nucleic acid ", as used herein, can also be described as a" nucleotide ", and has the same meaning and is defined by a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base and / or an analogue thereof, or a DNA or RNA polymerase (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)). If a modification to the nucleotide structure is present, such modification may be added before or after the synthesis of the nucleic acid amplimer. The nucleotide sequence may be terminated by a non-nucleotide component. Nucleic acid aptamers can be further modified after synthesis, e. G. By conjugation with a label.

The nucleic acid aptamers of the present invention are typically obtainable by in vitro selection methods for binding of target molecules. Methods for selecting platemers that specifically bind to target molecules are known in the art. For example, an organic molecule, a nucleotide, an amino acid, a polypeptide, a marker molecule on a cell surface, an ion, a metal, a salt, a polysaccharide may be a suitable target molecule for separating an umbrella that can specifically bind to each ligand . Selection of platemembers may utilize in vivo or in vitro selection techniques known in the art as SELEX (Systematic Evolution of Ligand by Exponential Enrichment) method (Ellington et al., Nature 346, 818-22, 1990; and Tuerk et al. , Science 249, 505-10, 1990). Specific methods for the selection and manufacture of platemers are described in U.S. Patent 5,582,981, WO 00/20040, U.S. Patent 5,270,163, Lorsch and Szostak, Biochemistry, 33: 973 (1994), Mannironi et al., Biochemistry 36: 9726 Blind, Proc. Natl. Acad. Sci. USA 96: 3606-3610 (1999), Huizenga and Szostak, Biochemistry, 34: 656-665 (1995), WO 99/54506, WO 99/27133, WO 97/42317 and U.S. Patent 5,756,291, Are incorporated herein by reference.

SELEX requires a single-stranded oligonucleotide pool or library consisting of randomized sequences. The term "oligonucleotide" in the present invention generally means a nucleic acid polymer comprising less than 100 nucleic acids. Oligonucleotides can be DNA, RNA or DNA / RNA hybrids that are not modified or modified. The oligonucleotide pool is a 100% random or partially random oligonucleotide, preferably the oligonucleotide pool can be composed of random or partially random oligonucleotides in which at least one fixed sequence and / or conserved sequence is included in the random sequence region have. More preferably, the oligonucleotide pool is a random or partially randomized sequence comprising at least one fixed and / or conserved sequence at which the 5 ' and / or 3 ' termini can consist of sequences shared in all molecules of the oligonucleotide pool Lt; / RTI > oligonucleotides. Oligonucleotide pools preferably contain not only random sequences but also fixed sequences that are essential for efficient replication. The oligonucleotide of the initial pool contains a fixed 5 ' and 3 ' end sequence, into which approximately 20-50 random nucleotides are inserted. In a specific embodiment of the present invention, a library in which 30 random nucleotides are inserted is used, and the nucleotide sequence length in the sequence amplification process can be changed. Random nucleotides can be produced by chemical synthesis and selection from randomly truncated intracellular nucleic acids.

The oligonucleotide may comprise a random sequence portion of a predetermined length and may be composed of a ribonucleotide and / or a deoxyribonucleotide, and may be a native nucleotide or a nucleotide analogue that has not been modified or modified (US Patent No. 5,958,691; No. 5,660,985, US Patent No. 5,958,691, US Patent No. 5,698, 687, US Patent No. 5,817, 635, US Patent No. 5,672,695, and PCT Publication WO 92/07065). Random oligonucleotides can be synthesized from phosphodiester-linked nucleotides using solid state oligonucleotide synthesis techniques well known in the art (Froehler et al., Nucl. Acid Res. 14: 5399-5467 (1986), Froehler et al., Tet. Lett., 27: 5575-5578 (1986)). Random oligonucleotides can also be synthesized using liquid phase methods such as the triester synthesis method (Sood et al., Nucl. Acid Res. 4: 2557 (1977), Hirose et al., Tet. Lett., 28 : 2449 (1978)).

In one embodiment of the invention, the nucleic acid overtamer of the invention consists of the sequence of SEQ ID NO: 9. It is also possible to use an RNA tymator sequence in which the thymidine nucleotide sequence of the DNA plasmid is substituted with uracil, Also, the nucleic acid aptamer of the present invention is interpreted to include a sequence that exhibits substantial identity with the sequence set forth in SEQ ID NO: 9. The above-mentioned substantial identity is determined by aligning the above-described sequence of the present invention with any other sequence as much as possible, and analyzing the aligned sequence using an algorithm commonly used in the art, at least 80% Homology, more preferably at least 90% homology, and most preferably at least 95% homology. Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described by Smith and Waterman, Adv. Appl. Math. 2: 482 (1981) Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is accessible from National Center for Biological Information (NBCI) It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLSAT is available at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

According to another aspect of the present invention, there is provided a composition for detecting bisphenol A comprising the above-described nucleic acid ablator. The composition for detecting bisphenol A of the present invention may contain carriers and / or preservatives, stabilizers, and the like known in the art for stable storage and preservation of nucleic acid platemers in addition to the above-described nucleic acid platemater.

In one embodiment of the invention, the nucleic acid aptamer of the present invention is attached with a detectable label. By attaching a detectable label to the nucleic acid platemer, it is possible to easily observe and measure the binding and the degree of binding between the target and the nucleic acid platemer. The detectable label may be a moiety that can be detected by a detection method known in the art, and is not particularly limited.

In one embodiment of the invention, the detectable label of the present invention is an optical label, an electrochemical label, a radioactive isotope, or a combination thereof. The label may be attached to a specific base or 3 ' or 5 ' end of the aptamer. The optical label may be, for example, a fluorescent substance. The fluorescent material may be selected from the group consisting of fluorescein, 6-FAM, rhodamine, Texas red, tetramethylrhodamine, carboxydodamine, carboxyrotamine 6G, carboxyrodone, carboxydodamine 110, cascade blue Cascade Blue), Cascade Yellow, Comarine, Cy2 (cyanine 2), Cy3, Cy3.5, Cy5, Cy5.5, Cy-chrome, Picoeritrin, PerCP (Peridinin chlorophyll- , PerCP-Cy5.5, JOE (6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein), NED, ROX (5- Hex, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Floor, 7-amino-4-methylcomarine- -Acetic acid, BODIPY FL, BODIPY FL-Br 2, BODIPY 530/550, conjugated cargoes thereof, and combinations thereof. For example, the fluorescent material may be fluorescein, Cy3 or Cy5. In addition, the optical label may include a fluorescent donor dye and a fluorescent acceptor dye separated by an appropriate distance, and the fluorescence emitted by the donor is fluorescence resonance energy transfer (FRET) pair . The donor pigments may include FAM, TAMRA, VIC, JOE, Cy3, Cy5 and Texas Red. The acceptor dye can be selected such that its excitation spectrum overlaps the donor emission spectrum. The receiver can also be a non-fluorescent receiver that quenches a wide range of donors. Other examples of donor-acceptor FRET pairs are known in the art. The electrochemical label includes an electrochemical label known in the art. For example, the electrochemical label may be methylene blue.

According to another aspect of the present invention, there is provided a bisphenol A detection kit comprising the above-described nucleic acid ablator.

The kit of the present invention can be prepared by using the above nucleic acid aptamer as a polymer compound of silica, semiconductor, plastic, gold, silver, magnetic molecule, nylon, poly (dimethylsiloxane), PDMS, cellulose, nitro Cellulose or a glass slide or the like. There is no particular restriction on the shape of the support, but may be in the form of a thin plate that can be held by hand, such as, for example, a glass slide, or in the form of a bead that is less than 0.1 mm in size, . The surface of the support may be functionalized with functional groups such as an aldehyde group, a carboxyl group, an epoxy group, an isothiocyanate group, an N-hydroxysuccinimidyl group, and an activated ester group, particularly an epoxy group. However, after the probe is fixed, it can be stabilized through a process of blocking the residual functional group to reduce the background signal.

The kits of the present invention may include buffer solutions and containers for performing and analyzing detection as needed, such as bottles, tubs, sachets, envelopes, tubes or ampoules, May take the same form and may be formed partly or wholly of plastic, glass, paper, foil or wax, and the like. The container may initially be a part of the container or may be fitted with a fully or partially detachable cap which may be attached to the container by mechanical, adhesive or other means. The container may also be equipped with a stopper accessible to the contents by the injection needle. The kit may include an external package, and the external package may include instructions for use of the components.

According to another aspect of the present invention, the present invention provides a bisphenol A detection method comprising the steps of:

(a) contacting the composition of any one of claims 3 to 5 with a sample of interest; And

(b) measuring the combined signal of the composition and the target sample after step (a).

The "object sample" of the present invention means an object to be subjected to the determination whether bisphenol A is detected or not, and is not particularly limited. Specific examples thereof include those obtained from polycarbonate plastic, epoxy resin, phenoplast resin, phenol resin, unsaturated polyester resin, thermal paper, polyol, modified polyamide, automobile tire or flame retardants and the like Can be used to detect bisphenol A, but are not limited thereto.

The binding signal measurement in step (b) of the present invention can be measured using a known method according to the characteristics of the detection signal used, and it is determined whether or not an increased signal is displayed as compared with the control group to confirm the presence of bisphenol A .

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a nucleic acid plasmid for detecting bisphenol A.

(b) The present invention provides a composition for detecting bisphenol A comprising the above-described nucleic acid ablator.

(c) The present invention provides a bisphenol A detection kit comprising the aforementioned nucleic acid platemater.

(d) The present invention provides a bisphenol A detection method using the composition for detecting bisphenol A described above.

(e) The nucleic acid aptamer of the present invention is structurally stable, has a long half-life, is easy to synthesize artificially, and can be easily chemically modified at each base sequence position, so that target substances can be detected using various methods.

(f) When bisphenol A detection nucleic acid plasmamer, composition, detection kit or detection method of the present invention is used, bisphenol A can be easily detected with high sensitivity.

1 shows a schematic diagram of the process of securing the bisphenol A in the DNA-BIND ^® 96-well plate. Bisphenol A was finally fixed on the surface coated with NOS functional group by using Tris and diepoxybutane as a linker.
Fig. 2 shows a schematic diagram of the SELEX process. From the original library, a pool of DNA was generated using the PCR method and the Crush and Soak method and bound to the fixed target material. In this process, single stranded DNA that is not bound to the target material was removed by a washing procedure. The single stranded DNA bound to the target substance was separated from the target material using a solution buffer and amplified using the PCR method. The amplified single stranded DNA was used as a library of the next round. The above procedure was referred to as a round, and the above procedure was repeated to confirm the sequence of the single strand DNA finally selected.
Fig. 3 shows the results of analysis of binding force (Kd) of platemers binding to bisphenol A. Fig. Biotin was labeled on the 5 'area of the selected platamer and the binding force was analyzed. The binding force (Kd) of platemer was confirmed to bind to bisphenol A at the concentration of nano molar concentration (nM).
Fig. 4 shows the results of the analysis of the specificity of the excavated compressors. Specificity was analyzed by the combination of bisphenol A (BPA), monoisononyl phthalate (MINP), penicillin G (PG), and DEB (diepoxybutane) It was confirmed that the aptamer of the present invention has a better binding force to bisphenol A.
5 shows a table summarizing selection conditions for SELEX execution. From Step 1 to Step 10, the total salt and DMSO concentration, the type of target substance, and the library binding time were adjusted to separate potent and specific binding proteins. Counter selection was carried out using nonylphenol, bisphenol AF and 4-phenylphenol structurally similar to bisphenol A to increase the specificity of the platamer.
Figure 6 shows platelet sequences that specifically bind to bisphenol A. After the end of SELEX in 10 steps, 30 samples were sequenced and 9 kinds of sequences were selected. Especially, the frequency of platamer of the 9th sequence was the highest.

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 embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Fixation of Bisphenol A for single strand DNA extramammary screening (SELEX)

In order to screen the aptamer specifically binding to the bisphenol A was first conducted a process of fixing the above DNA-BIND ^® 96-well plate by the use of bisphenol A diepoxide Tris and retain part (diepoxybutane). DNA-BIND ^® 96-well plate is the well surface-activated with N- oxy succinimide (oxysuccinimide) functional groups covalently bonded to it to form the amine functional group to react at pH 8.5 and conditions. Based on this principle, Tris is fixed and used as a primary linker. To fix the Tris, 200 μl of Tris-HCl buffer (1 M, pH 8.5) was added to the wells of the plate and reacted at room temperature for 1 hour. After washing 5 times, diepoxybutane (3 M, pH 13) was reacted with the hydroxy functional group of Tris attached to the plate for 4 hours at 37 ° C. After washing 7 times, bisphenol A (50% DMF, 1.5 M, pH 13) was reacted with the epoxide functional group at 37 ° C for 4 hours (see FIG. 1).

Example 2: Single stranded DNA DNA typing that specifically binds to bisphenol A (SELEX)

Single stranded DNA extramammers capable of specifically binding to the environmental hormone bisphenol A were selected from the random single stranded DNA library (Bioneer, Deajeon, Korea) consisting of approximately 7.2 x 10 ¹⁴ different DNA sequences by the SELEX method Respectively. The random DNA library is first required to find a specific binding pressure-timer using the bisphenol A immobilized in Example 1 above. For this purpose, a single stranded DNA library (5'-ATGCGGATCCCGCGC (N) ₃₀ GCGCGAAGCTTGCGC-3 ') containing ₃₀ randomly generated nucleotide sequences (N) ₃₀ was used in a total of 60 bases. 5'-ATGCGGATCCCGCGC-3 '(containing BamHI site) and the reverse primer'5'-GCGCAAGCTTCGCGC-3'(including HindIII site) were used to amplify a template DNA library. Were synthesized and synthesized. The template DNA library was amplified by asymmetric PCR using 100 μM forward primer and 10 μM reverse primer to obtain single-stranded DNA only. The PCR product was electrophoresed on 2.5% agarose gel to confirm the product. After PCR, Crush and Soak method was used to isolate single stranded DNA library. The PCR product was electrophoresed on a 12% native gel to separate double strand and single strand DNA. After electrophoresis, DNA was stained with EtBr, and single-stranded DNA bands were cut. The cut gel was pulverized and leached single-stranded DNA using Crush and Soak buffer (500 mM NH ₄ OAc, 0.1% SDS, 0.1 mM EDTA) . The solids were centrifuged and the solid gel was separated and purified by ethanol precipitation and then quantified using a UV spectrophotometer and used for SELEX.

SELEX, an in vitro selection of oligonucleotides, was performed to screen platemers that specifically bind to bisphenol A using single stranded DNA. After fixed bisphenol A and platemer were reacted, the non-bonded platamer was removed and the weak platamer was also removed through the washing process. A high pH elution buffer (40 mM Tris, 10 mM EDTA, 10 mM NaOH) was used to isolate the tyramine bound to bisphenol A. In the high pH condition, since the structure of the extruder is deformed, it is eluted from the bisphenol A. The eluted platamer was amplified by PCR, and only the platamer was obtained through 12% native DNA electrophoresis and the next round was performed (see FIG. 2). In this case, when binding bisphenol A with ssDNA, the ssDNA expected to bind to bisphenol A with high binding force under extreme conditions (see FIG. 5) Respectively.

6, 8, and 10 rounds, ssDNA binding to bisphenol A-like structures (bisphenol AF, phenylphenol, nonylphenol) was removed to minimize non-specific binding of ssDNA. Instead of bisphenol A, a bisphenol A-like structure was added to isolate ssDNA that was not bound to the bisphenol A-like structure, which was then used in the next step to increase the specificity of the excised tympanum.

Cloning was performed to analyze the sequence of ssDNA obtained through SELEX process in the final 10 rounds. The ssDNA was amplified with dsDNA using primers of the same concentration and inserted into pET 28a vector, a vector for bacterial expression having a T7 promoter for sequencing. BamHI and Hind III were selected as restriction enzymes, respectively. Recombinant plasmids were obtained by using DNA ligase for insertion of gene sequence into expression vector. A total of 10 screening and amplification steps for bisphenol A were performed to finally select one ssDNA compactor. The nucleotide sequences of these plasmids are shown in Fig. 6 below.

Example 3: Analysis of the binding force between bisphenol A and one plastomer sequence synthesized

The embodiment 1 kinds Example 2 sequencing enzyme immunoassay based on the aptamer to determine their binding affinity to bisphenol A found in using (Aptamer based ELISA) showing a higher high frequency is also of aptamer binding to bisphenol A (5'-ATGCGGATCCCGCGCCGAGTGAAGGCAAGGCGCGGCGTCCCTTCGGTCCGCGCGAAGCTTGCGC-3 ') . For the measurement, biotin was connected to the 5 'end. The amount of platamer bound to bisphenol A was measured by binding of biotin and streptavidin to diluted platemaker with a plate (96 well plate, SPL) on which bisphenol A was immobilized. More specifically, bisphenol A, the DNA-BIND ^® the embodiment a 96-well plate were fixed by using a linker as described in Example 1, aptamer binding buffer (25 mM Tris, 100 mM NaCl , 25 mM KCl, 2, 5 mM MgCl ₂ , 5% DMSO in dH ₂ O, pH 8.0), followed by addition of platamer for binding reaction with bisphenol A for 1 hour. Thereafter, the cells were washed 10 times with 1X PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ 0.1% Tween 20 in dH ₂ O, pH 8.5) , And streptavidin (HRP) were reacted for 1 hour. After washing 10 times with washing solution, TMB substrate solution (3,3 ', 5'-tetramethylbenzidine (TMB) / H ₂ O ₂ , Chemicon) was added and after 15 minutes, 1 M sulfuric acid was added to terminate the reaction. The whole process was named ELISA (enzyme-linked immunosorbant assay) using platemer and the K _d (dissociation constant) value obtained as a result of this experiment was determined by ELISA The saturation curve is determined by measuring the signal intensity at 450 nm. As shown in FIG. 3, the aptamer according to the present invention shows a binding force at a level of nano molarity (nM).

Example  4: One synthesized Abtamer  Sequence specificity analysis

In order to investigate the specificity of the nucleotide sequence found in Example 2 above, platamer and bisphenol A (BPA), monoisononyl phthalate (MINP), and penicillin extracted using an Aptamer-based ELISA G (PG), and diepoxybutane (DEB). Bisphenol A is a substance widely used as a plasticizer together with phthalates. Therefore, we have selected MINP, one of the phthalates, as the target substance of the specificity analysis to confirm that the two can be distinguished practically. Penicillins have various ring structure functional groups in structure. Among them, penicillin G has a benzene ring, which is structurally similar to the hydroxyphenyl functional group of bisphenol A. Penicillin G was also used as the target of the specificity analysis. For the measurement, biotin was connected to the 5 'terminus. Bisphenol A is a fixed plate ^{(DNA-BIND ® 96 well plate} , Corning) to put the diluted aptamer concentration was measured by the amount of aptamer bound using a combination of biotin and streptavidin. More specifically, bisphenol A, the DNA-BIND ^® the embodiment a 96-well plate were fixed by using a linker as described in Example 1 into the aptamer rear semi washed 5 times with aptamer binding buffer Bisphenol for 1 hour A reaction. Thereafter, the cells were washed 10 times with 1X PBST as a washing solution, and streptavidin (HRP) was reacted for 1 hour. After rinsing 10 times with washing solution, TMB substrate solution was added and after 15 minutes, 1 M sulfuric acid was added to terminate the reaction. The ELISA signal intensity according to the corresponding concentration of the platemor reacted to the fixed bisphenol A-like structure of each well is determined by the saturation curve as measured at the absorbance at a wavelength of 450 nm. As shown in FIG. 4, it was confirmed that the aptamer according to the present invention specifically binds to bisphenol A.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> IUCF-HYU (Industry-University Cooperation Foundation Hanyang University) <120> Nucleotide Aptamer for Use in Detection of Bisphenol A <130> PN160089 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Bisphenol A binding aptamer <400> 1 atgcggatcc cgcgccgacg aaggaaggcg cacgacctca cggccgcgcg aagcttgcgc 60                                                                           60 <210> 2 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Bisphenol A binding aptamer <400> 2 atgcggatcc cgcgcacgag ggccaggtac agacacgggt tgccggcgcg aagcttgcgc 60                                                                           60 <210> 3 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Bisphenol A binding aptamer <400> 3 atgcggatcc cgcgccatgg tgggccttga cagccgggtg gcgcggcgcg aagcttgcgc 60                                                                           60 <210> 4 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Bisphenol A binding aptamer <400> 4 atgcggatcc cgcgccggag ggaggccgcc gacggaccgg gtgcggcgcg aagcttgcgc 60                                                                           60 <210> 5 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Bisphenol A binding aptamer <400> 5 atgcggatcc cgcgccgccg atggggtggc gcgcttccgt tggtcgcgcg aagcttgcgc 60                                                                           60 <210> 6 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Bisphenol A binding aptamer <400> 6 atgcggatcc cgcgcagtgg ggcagaccag ccgggtatcc gggtagcgcg aagcttgcgc 60                                                                           60 <210> 7 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Bisphenol A binding aptamer <400> 7 atgcggatcc cgcgcagtgg gggatgccgt gcatgcgtgg ggcgggcgcg aagcttgcgc 60                                                                           60 <210> 8 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Bisphenol A binding aptamer <400> 8 atgcggatcc cgcgcagggc gaggcgagct aggtgatcga cctacgcgcg aagcttgcgc 60                                                                           60 <210> 9 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Bisphenol A binding aptamer <400> 9 atgcggatcc cgcgccgagt gaaggcaagg cgcggcgtcc cttcggtccg cgcgaagctt 60 gcgc 64

Claims (7)

Nucleic acid plasmids for the detection of bisphenol A comprising sequence 9 of Sequence Listing.
2. The nucleic acid plasmid according to claim 1, wherein the nucleic acid aptamer comprises the sequence of SEQ ID NO: 9.
A composition for detecting bisphenol A, comprising the nucleic acid ablator of claim 1.
4. The composition of claim 3, wherein the nucleic acid aptamer is attached with a detectable label.
5. The composition of claim 4, wherein the detectable label is an optical label, an electrochemical label, a radioisotope, or a combination thereof.
A bisphenol A detection kit comprising the nucleic acid abstamator of claim 1.
A bisphenol A detection method comprising the steps of:
(a) contacting the composition of any one of claims 3 to 5 with a sample of interest; And
(b) measuring the combined signal of the composition and the target sample after step (a).

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