KR20180089974A - Single strand DNA aptamer for detection of phthalate - Google Patents

Abstract

The present invention relates to a single-stranded DNA aptamer which specifically binds to phthalate, and a kit and a composition for detecting phthalate using the same. According to the present invention, the single-stranded DNA aptamer has high binding force and specificity to phthalate, which is a kind of endocrine disrupters and used as a plasticizer in industries, and it is possible to detect phthalate more sensitively and accurately than conventionally used analytic methods.

Description

Single strand DNA aptamer for detection of phthalate which specifically binds to phthalate.

The present invention relates to a single-stranded DNA plasmid that specifically binds to a phthalate, and a kit and a composition for detecting phthalate using the same.

"Phthalate", which is used as a plasticizer, is a type of endocrine disrupter that interferes with or disrupts the action of hormones in animals or people. Phthalates have toxicity comparable to that of cadmium and are toxic substances that are toxic to reproductive organs due to infertility in female, infertility and decreased number of sperm, which have negative effects on liver, kidney, heart and lungs.

Europe, the United States and other countries have concluded that phthalate plasticizers are harmful to human body and classify them as harmful substance group and broaden the prohibition of use of toys and infants. In 2002, Dagensnietteji of Sweden had phthalate in fragrance After reporting, the EU prevented DEHP and DBP from being used in cosmetics.

Environmental hormones, unlike biohormones, are difficult to decompose, remain in the ecosystem circulation for a long time, and cause disorders in the living body. Domestic environmental hormone consumption / output will continue to increase given the increasing size of the economy and rising consumption levels, and the risk of exposures to hazardous chemicals is increasing due to the frequent movement of chemicals across countries. The technology available is extremely limited.

Currently, chromatographic methods such as HPLC (High Performance Liquid Chromatography) and LC-MS (Liquid Chromatography with Tandem Mass Spectrometry) are commonly used methods for detecting phthalates.

However, this method is advantageous in that it can be quantitated to a very low concentration because of its high specificity, high sensitivity, and selectivity. However, since it requires expensive equipment for analysis, it is difficult to detect it quickly in the field. In addition, since a large-sized machine is used, it can not be carried, its analysis time is long, and it is costly.

Another method for detecting low molecular weight compounds such as phthalates is biosensor technology using antibodies. However, the diagnostic biosensor using the antibody has a disadvantage in that it is difficult to produce the antibody due to the antibody having a large molecular structure, and the antibody is not easily deformed. In addition, since antibodies are produced using animals or cells, they can be very time-consuming and costly to mass-produce, and their functionality can vary depending on the timing of production, thus greatly affecting the accuracy of antibody-based biosensors. In addition, since the antibody is difficult to be stored or transported at room temperature, there is a problem in using the antibody as a diagnostic biosensor requiring a long time or repeated use.

Therefore, there is a need for a new assay that can detect phthalates more sensitively and accurately by introducing molecular recognition tools that can replace antibodies.

The present inventors have found that by studying a single-stranded nucleic acid (DNA, RNA), Aptamer, which has a stable tertiary structure per se and is capable of binding to a target molecule with high affinity and specificity, Single strand DNA extramamers capable of detecting phthalates, one of the materials, were unearthed.

It is an object of the present invention to provide a single-stranded DNA plasmid that specifically binds phthalates.

Another object of the present invention is to provide a composition and a kit for detecting phthalate comprising the single-stranded DNA extruder.

In order to achieve the above object, the present invention provides a single-stranded DNA extramammer that specifically binds to a phthalate comprising any one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 .

The present invention also provides a composition for detecting phthalate comprising the single-stranded DNA compact.

The present invention also provides a phthalate detection kit comprising the single-stranded DNA extruder.

Preferably, the phthalate may be diethylhexyl phthalate (DEHP) or monoethylhexyl phthalate (MEHP).

The present invention also relates to a method for preparing EDA, comprising the steps of (a) coating a plate with NOS (N-oxysuccinimide) group, (b) fixing EDA to the plate surface by reacting EDA (Ethylenediamine) (D) a step of reacting the reactant of step (c) with a plate on which the EDA is immobilized; and (c) Wherein the phthalate is immobilized on the NOS coated plate by immobilizing the phthalate on the NOS coated plate.

Also, the present invention provides a method for manufacturing a semiconductor device, comprising: (a) fixing a phthalate to an NOS (N-oxysuccinimide) coating plate by the immobilization method; (b) reacting a single-stranded DNA extramamer expected to specifically bind to the phthalate with the phthalate-immobilized NOS-coated plate; and (c) separating the platemater bound to the phthalate. The present invention also provides a method of screening a single stranded DNA DNA tymator that specifically binds phthalate.

The single stranded DNA aptamer of the present invention can detect phthalates more sensitively and accurately than conventional methods because it has high binding force and specificity to phthalates which are a kind of environmental hormones and used as a plasticizer in the industrial field. In addition, it is advantageous in that it can be used as a biosensor or kit for phthalate detection because it is less in production cost than an antibody and is easy to immobilize on a surface. The single stranded DNA aptamer of the present invention can be developed not only as an environmental hormone but also as a diagnostic agent for environmentally harmful substances such as residual antibiotics and heavy metals.

Figure 1 shows the structural formulas of various types of phthalates.
2 is a schematic view showing a method of immobilizing phthalate on a plate coated with NOS (N-oxysuccinimide).
FIG. 3 is a graph showing the binding strength of single-stranded DNA extruder to MEHP (monoethylhexyl phthalate) according to the present invention.
FIG. 4 is a graph showing the specificity of single stranded DNA extender for MEHP (monoethylhexyl phthalate) according to the present invention.
5 is a graph showing the binding strength of single stranded DNA extender according to the present invention to DEHP (Diethylhexyl phthalate).
FIG. 6 is a graph showing the specificity of single stranded DNA extender for DEHP (Diethylhexyl phthalate) 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.

In one aspect, the present invention relates to a single stranded DNA plasmid that specifically binds to a phthalate comprising at least one base sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.

"Aptamer" is a low molecular probe that can be used as a single-stranded nucleic acid (DNA or RNA) of short length (20 to 60 nucleotides), capable of binding with high affinity and specificity to various kinds of target ligands, ) Piece.

Although aptamers have properties similar to those of antibodies in that they have high binding and selectivity at the nanomolar-picomolar level for target substances, they are easier to chemically synthesize than antibodies and can be stored for a long time at room temperature, It has an advantage that it hardly causes an immune response.

In general, the development of platamers is carried out in vitro using the method called "Systematic Evolution of Ligands by Exponential Enrichment" (SELEX).

The central principle of SELEX is that only individuals with a certain trait will survive from a large number of randomly existing individuals, and the survival of individuals will gradually increase in their proportion, and finally, Very similar. However, the individual in SELEX is a single-stranded nucleic acid, and the final choice is made based on the binding capacity of the platamer to a specific ligand. Therefore, it is necessary to first prepare a large number of nucleic acid libraries with random sequences, to isolate individuals with high binding capacity for specific ligands that are expected to be present in this library, Amplification to increase the ratio in the nucleic acid pool, and amplification so that selection of the next round can proceed.

Generally, a process of obtaining a single-stranded DNA pool from a library having 10 ¹⁴ to 10 ¹⁵ different sequences, that is, diversity is required. As a method for this, there is a method of amplifying only one strand using asymmetric PCR, a method of attaching biotin to a strand end of double strand DNA and selectively isolating one strand using bead wrapped with streptavidin.

The resulting library is then ligated to the target ligand, and a selection process is performed to select the plasmids with high affinity. When the target substance is a low-molecular substance, it is usually immobilized on a bead or a resin through a covalent bond, and then an affinity column is formed using the immobilized substance. A nucleic acid library is flowed through the column to induce binding, followed by washing with a buffer to remove nucleic acids that do not bind to the ligand. The platamers bound to the ligand immobilized on the column are washed off with a solution containing a low-salt buffer or ligand. When the target substance is a protein, usually a nitrocellulose filer is used to separate the platamer bound to the protein. These methods can be used to obtain platamers having affinity for the ligand. Repeating 5-15 cycles of sorting and amplification usually yields a high affinity potentiometer. When the selection process is completed, the amplified DNA is cloned, the sequence is identified from the individual clones, and the affirmator is synthesized to measure the affinity with the target substance and the binding force.

The inventors of the present invention conducted screening of a single nucleic acid platamer binding to a fixed phthalate with high affinity and specificity by newly attempting a method of covalently binding phthalate, which is a low molecular substance, on a plate surface to detect phthalate having high binding force and specificity Stranded DNA strand was extracted.

At this time, the phthalate immobilization method includes (a) coating NOS (N-oxysuccinimide) group on a plate; (b) immobilizing EDA on the plate surface by reacting EDA (Ethylenediamine) on the coated plate; (c) sequentially reacting phthalate with EDC (1-Ethyl-3- (3-dimethylaminopropyl) -carbodiimide) and NHS (N-Hydroxysuccinimide); (d) fixing the phthalate to the NOS-coated plate by reacting the reactant of step (c) with a plate on which the EDA is immobilized.

In one embodiment of the present invention, EDA (Ethylenediamine) was immobilized on the surface of the plate and used as a primary linker. The carboxyl group and amine of the target phthalate were connected to each other through an EDC / NHS reaction, Was fixed on the plate.

As described above, the single-stranded DNA extramammary that specifically binds to the phthalate can be screened by fixing the phthalate to the NOS coated plate. The method comprises (a) preparing a NOS (N-oxysuccinimide) coated plate Immobilizing a phthalate on the substrate; (b) reacting a single-stranded DNA extramamer expected to specifically bind to the phthalate with the phthalate-immobilized NOS-coated plate; (c) separating the platemer bound to the phthalate.

In one embodiment of the present invention, monoethylhexylphthalate (MEHP) was immobilized on a plate coated with NOS (N-oxysuccinimide) group, and a single-stranded DNA extender capable of specifically binding to MEHP was selected by the SELEX method. A total of 10 single-stranded DNA extramammers were screened and analyzed for binding force using an enzyme immunoassay (Aptamer based ELISA). As a result, single-stranded DNA aptamers of SEQ ID NO: 1 and SEQ ID NO: 2 specifically bound to phthalate Respectively.

Examples of the phthalates include di- (2-ethylhexyl) phthalate (DEHP), mono (2-ethylhexyl) phthalate (MEHP), monononyl phthalate Mono-nonyl phthalate (MINP), and monobenzyl phthalate (MBP). In addition, butyl benzyl phthalate (BBP), di-n-butyl phthalate (DBP), diethyl phthalate Diethyl phthalate (DEP), dimethylphthalate (DMP), di-hexylphthalate (DHP), dipropyl phthalate (DprP), dicyclohexyl phthalate (DCHP) Di-n-pentyl phthalate (DPP), di-iso-nonylphthalate (DINP), di-n-octylphalate (DNOP), di- iso-decylphthalate , DIDP). Among them, DEHP Accounting for a quarter of a plasticizer.

In the present invention, the phthalate may be diethylhexyl phthalate (DEHP) or monoethylhexyl phthalate (MEHP), and more preferably diethylhexyl phthalate (DEHP).

In another aspect, the present invention relates to a composition for detecting phthalate comprising the single stranded DNA compact.

Since the single stranded DNA aptamer according to the present invention can specifically detect only phthalate, after contacting the sample suspected of containing the phthalate with the composition and confirming whether the phthalate and the single stranded DNA strand are bound to each other , And a method of detecting phthalate.

Furthermore, the single strand DNA extender may be used to separate or remove the phthalate from a specific sample. For example, phthalates can be separated or removed from a specific sample by filling the column with the fixed beads and passing the sample containing the phthalate.

Here, the sample may be a sample collected from at least one of water, soil, wastes, food, plants and animals, and animal and plant tissues, but the present invention is not limited thereto. At this time, the water may include precipitation, seawater, lake and storm, and the waste may include sewage, wastewater, etc., and the animal or plant may include a human body.

In another aspect, the present invention relates to a phthalate detection kit comprising the single stranded DNA compact.

The single stranded DNA aptamer of the present invention can be immobilized on the surface, and its production cost is smaller than that of the antibody, so that it is easy to develop a kit for detecting phthalate.

The phthalate detection kit may take the form of a bottle, a barrel, a small bag, an envelope, a tube, an ampoule, etc., which may be formed partly or wholly of plastic, glass, paper, foil or wax. The kit may include an external package, and the external package may include instructions for use of the components.

In addition, it can be developed as a biosensor such as a phthalate detection chip as well as a phthalate detection kit including the single strand DNA extramammary.

Since the single stranded DNA aptamer of the present invention has a lower production cost than an antibody and can be easily immobilized on a surface thereof, it is more sensitive and accurate than a conventional antibody-based analysis method in developing a biosensor such as a phthalate detection kit or a chip, Can be detected.

In particular, phthalates are a kind of environmental hormones that can affect the human body through various environmental pathways when they are present in the final production of plastic products or cosmetics as well as in the manufacturing process of such products. Therefore, the single stranded DNA extruder according to the present invention can be used to carry out inspections for the presence or absence of phthalates in the production of plastics and cosmetics.

Furthermore, the single stranded DNA aptamer of the present invention can be developed not only as an environmental hormone but also as a diagnostic agent for environmentally harmful substances such as residual antibiotics and heavy metals.

Example

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

Example  One : Single strand  DNA Abtamer  For screening Phthalate  fixing

For phthalate-specific binding pressure screening, MEHP (monoethylhexylphthalate) was fixed on a plate coated with NOS (N-oxysuccinimide) group. The NOS (N-oxysuccinimide) group is a specific functional group that forms an amide bond by reacting with a primary amine under weakly alkaline conditions. Based on this principle, EDA (Ethylenediamine) was immobilized on the plate surface and used as a primary linker. To fix EDA, 200 μl of 1M EDA was reacted at room temperature for 1 hour. After washing 5 times with 1X PBST (composition), the MEHP was fixed by EDC / NHS reaction. The EDC / NHS reaction was carried out by reacting 40 mM MEHP, 100 mM EDC and 250 mM NHS for 15 minutes and then reacting on EDA-immobilized NOS coated plate at room temperature for 2 hours and 30 minutes. And washed 10 times with 1X PBST (Fig. 2).

Example  2 : Phthalate  Specific binding Single strand  DNA Abtamer  Screening ( SELEX )

Single-stranded DNA extramammers capable of specifically binding to MEHP, one of the phthalate classes, from a random single-stranded DNA library (Bioneer, Deajeon, Korea) consisting of approximately 7.2 x 10 ¹⁴ different DNA sequences were prepared by SELEX method Respectively.

A random DNA library was constructed in order to extract the specific binding pressure thermometer using the immobilized MEHP in Example 1 above. A single stranded DNA library (5'-ATGCGGATCCCGCGC (N) ₃₀ GCGCGAAGCTTGCGC-3 ') containing ₃₀ randomly constructed nucleotide sequences ((N) ₃₀ ) in a total of 60 nucleotide sequences was used.

5'-ATGCGGATCCCGCGC-3 '(containing BamHI site) and 5'-GCGCAAGCTTCGCGC-3' (containing HindIII sites)] to amplify a template DNA library Respectively. The template DNA library was amplified by asymmetric PCR using 100 uM forward primer and 10 uM reverse primer to obtain single strand 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 single-stranded DNA was extracted with Crush and Soak buffer (500 mM NH ₄ OAc, 0.1% SDS, 0.1 mM EDTA). The leachate was centrifuged, and the solid gel was separated and purified by ethanol precipitation and then quantified using UV Spectrophotometer and used for SELEX.

In Vitro selection of oligonucleotides, SELEX, was performed to screen platemers that specifically bind to MEHP using single stranded DNA. After the fixed phthalate was reacted with the platemer, the uncombined 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 separate the platamer bound to the phthalate. At high pH conditions, the structure of the pressure tampers is deformed and thus separated from the phthalates. The separated platamer was amplified by PCR, and only the platamer was obtained by 12% native DNA electrophoresis and the next round was performed. At this time, when binding phthalate with ssDNA, ssDNA expected to bind to phthalate with high binding force even under the extreme conditions (Table 1) was obtained through binding conditions that change the target substance and binding time according to the buffer condition.

SELEX  process Round One 2 3 4 5 6 7 8 ssDNA 200
Pmol Binding Buffer 1X 1X 1.5X 2X 2X 2X 2X 2.5X Incubation time 90 60 60 45 45 45 30 30 Negative
selection - MINP MINP -

(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)

In order to minimize the non-specific binding of ssDNA, ssDNA binding with Mono-isononyl phthalate (MINP), another kind of phthalate, was removed in the 5th and 6th rounds. The ssDNA not bound to MINP was isolated and used in the next step to increase the specificity of the excised tympanum.

The cloning procedure was performed to analyze the sequence of ssDNA obtained through the SELEX process in the final 8 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 HindIII were selected for restriction enzymes, respectively. Recombinant plasmids were obtained by using DNA ligase for insertion of gene sequences into expression vectors. Finally, 10 ssDNA compactor sequences were selected through a total of 8 screening and amplification steps for phthalates. The nucleotide sequences of these plasmids are shown in Table 2 below.

SEQ ID NO: order Poverty
(Frequency) One ATG CGG ATC CCG CGC GAC CAA CGG AAG CGC GGC ACC ACA ACG GTG GCG CGA AGC TTG CGC 21 2 ATG CGG ATC CCG CGC GGG TCT GAG GAG TGC GCG GTG CCA GTG AGT GCG CGA AGC TTG CGC 4 3 ATG CGG ATC CCG CGC GGA CAG CGG GGC TGC TCC ATA CTG CAT GTG GCG CGA AGC TTG CGC 3 4 ATG CGG ATC CCG CGC CGC ATA GCA GTG GGG GAC GTT TCC TGC ATG GCG CGA AGC TTG CGC 2 5 ATG CGG ATC CCG CGC GGG GAC GGT GCT GCG TCC TTT GGG GGG GTG GCG CGA AGC TTG CGC One 6 ATG CGG ATC CCG CGC GCA CGG TGG ACA ACG CAC GCC TGC ATA CAC GCG CGA AGC TTG CGC One 7 ATG CGG ATC CCG CGC CGT CGA AGG GTG CGT CCA TGA CCG TGT GCG CGA AGC TTG CGC One 8 ATG CGG ATC CCG CGC TGG ACG AGG TGC CGT GTT CCC CTC CTA TCG GCG CGA AGC TTG CGC One 9 ATG CGG ATC CCG CGC GAA TGG GCG GAA TCG CAG GAG GGA GTC ACT GCG CGA AGC TTG CGC One 10 ATG CGG ATC CCG CGC TGG GCA CCG GGC AGT CGG GGT TGC AAG AGT GCG CGA AGC TTG CGC One

Example  3: Abtamer  Sequence analysis and binding assay

To determine the binding ability of the nucleotide sequence found in Example 2 to the phthalate, aptamer based ELISA was used to determine the binding capacity of phthalates to phthalates, '- ATG CGG ATC CCG CGC GAC CAA CGG AAG CGC GGC ACC ACA ACG GTG GCG CGA AGC TTG CGC - 3'), (5 '- ATG CGG ATC CCG CGC GGG TCT GAG GAG TGC GCG GTG CCA GTG AGT GCG CGA AGC TTG CGC - 3 '). Biotin was connected to the 5 'terminus for binding assay. The amount of platamer bound to the target substance was measured using a combination of biotin and streptavidin by diluting the platemaker with concentration of phthalate in a plate (96 well plate, SPL) on which phthalate was immobilized. More specifically, phthalates were fixed on NOS coated plates using a linker as described in Example 1, and then platemater 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 and binding reaction with phthalate for 2 hours. 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 is called an enzyme-linked immunosorbant assay (ELISA) using platemer, and the resultant K _d The dissociation constant value is defined as the saturation curve, measured at 450 nm, of the ELISA signal intensity according to the corresponding concentration of the platemorant reacted with the immobilized protein in each well. As a result, it was confirmed that the binding force of the tympanic membrane of SEQ ID NO: 1 was 62.71 nM and the binding force of the tympanic membrane of SEQ ID NO: 2 was 196.06 nM, indicating that the aptamer according to the present invention had a binding strength of nano molarity (Fig. 3).

Example  4: Synthesized To Abtaram  Sequence specificity analysis for

In order to investigate the specificity of the nucleotide sequence found in Example 2, a digestor digested with an aptamer based ELISA, MEHP, monoisononyl phthalate (MINP), and monobenzyl phthalate (MBP ) Were analyzed for specificity. Specificity was measured using different kinds of phthalates to confirm the specificity of the base sequence. Phthalates are frequently used as plasticizers together with phthalates. Therefore, in order to make sure that the two can be distinguished from each other, MINP and MBP were selected as the phthalates as the target of the specificity analysis.

To analyze the specificity, the above phthalates were fixed on a NOS-coated plate using a linker as described in Example 1, and then incubated with a platamater binding buffer (25 mM Tris, 100 mM NaCl, 25 mM KCl, mM MgCl ₂ , 5% DMSO in dH ₂ O, pH 8.0) for 5 hours, and then added to the platamer for binding reaction with phthalates for 1 hour. Thereafter, washing solution was subjected to 10 washing steps using 1X PBST, and streptavidin (HRP) was reacted for 1 hour. After rinsing 10 times with washing solution, TMB substrate solution was added and after 15 minutes, 1M sulfuric acid was added to terminate the reaction. The ELISA signal intensity according to the corresponding concentrations of the platelets reacted with the fixed phthalates of each well is determined by the saturation curve measured from the absorbance at 450 nm wavelength.

As a result, it was confirmed that both the platemer of SEQ ID NO: 1 and the platemer of SEQ ID NO: 2 specifically bind to MEHP (FIG. 4).

Example  5: Reduction Graphene oxide  Analysis of binding force for DEHP using

(5 '- ATG CGG ATC CCG GAC CAA CGG AAG CGC GGC ACC ACA ACG GTG GCG CGA AGC TTG CGC - 3'), which combines high binding force and specificity to MEHP, The binding force to DEHP was analyzed for CGC GGG TCT GAG GAG TGC GCG GTG CCA GTG AGT GCG CGA AGC TTG CGC - 3 '). When the MEHP is immobilized on a NOS coated plate, the exposed MEHP matches the DEHP, so that the DEHP detection of the above MEHP bound aptamer is possible.

In the case of reduced graphene oxide, it has a variety of functional groups and can be bonded to various molecules according to surface functionalization. Pyrene, which is a unit of graphene oxide, has an aromatic property, . It can also cause FRET phenomenon that acts as an electron acceptor and loses fluorescence depending on the distance from the fluorescent molecule. Based on these properties, FAM (6-Carboxyfluorescein) was synthesized by linking each tablet.

The fluorescence intensity of recovered fluorescence was measured by treating 4 ug / ul of reduced graphene oxide with a concentration range of 0 to 4 uM and reducing the fluorescence by treatment with phthalate (1 mM). The binding force was analyzed. As a result, it was confirmed that the binding force of the tympanic membrane of SEQ ID NO: 1 was 235.3 nM and that of the tympanic membrane of SEQ ID NO: 2 was 237.9 nM (FIG. 5).

Example  6: Reduction Graphene oxide  Specificity of DEHP used

In order to analyze the specificity of the tympanic membrane, DEHP, MINP and MBP were tested in the same manner as in Example 5 above. As a result of the detection of the specificity of DEHP using reduced graphene oxide, as in the results of the MEHP specificity analysis using the ELISA method, both the platemer of SEQ ID NO: 1 and the platemer of SEQ ID NO: 2 specifically bind to DEHP (Fig. 6).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Industry-University Cooperation Foundation Hanyang University <120> Single strand DNA aptamer for detection of phthalate <130> WPN17001 <160> 10 <170> KoPatentin 3.0 <210> 1 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Aptamer 1 <400> 1 atgcggatcc cgcgcgacca acggaagcgc ggcaccacaa cggtggcgcg aagcttgcgc 60                                                                           60 <210> 2 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Aptamer 2 <400> 2 atgcggatcc cgcgcgggtc tgaggagtgc gcggtgccag tgagtgcgcg aagcttgcgc 60                                                                           60 <210> 3 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Aptamer 3 <400> 3 atgcggatcc cgcgcggaca acggggctgc tccatactgc atgtggcgcg aagcttgcgc 60                                                                           60 <210> 4 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Aptamer 4 <400> 4 atgcggatcc cgcgccgcat agcagtgggg gacgtttcct gcatggcgcg aagcttgcgc 60                                                                           60 <210> 5 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Aptamer 5 <400> 5 atgcggatcc cgcgcgggga cggtgctgcg tcctttgggg gggtggcgcg aagcttgcgc 60                                                                           60 <210> 6 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Aptamer 6 <400> 6 atgcggatcc cgcgcgcacg gtggacaacg cacgcctgca tacacgcgcg aagcttgcgc 60                                                                           60 <210> 7 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Aptamer 7 <400> 7 atgcggatcc cgcgccgtcg aagggtgcgt ccatgaccgt gtgcgcgaag cttgcgc 57 <210> 8 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Aptamer 8 <400> 8 atgcggatcc cgcgctggac gaggtgccgt gttcccctcc tatcggcgcg aagcttgcgc 60                                                                           60 <210> 9 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Aptamer 9 <400> 9 atgcggatcc cgcgcgaatg ggcggaatcg caggagggag tcactgcgcg aagcttgcgc 60                                                                           60 <210> 10 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Aptamer 10 <400> 10 atgcggatcc cgcgctgggc accgggcagt cggggttgca agagtgcgcg aagcttgcgc 60                                                                           60

Claims (8)

A single stranded DNA plasmid that specifically binds to a phthalate comprising at least one nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
The single stranded DNA extramammary according to claim 1, wherein the phthalate is diethylhexyl phthalate (DEHP) or monoethylhexyl phthalate (MEHP).
A composition for detecting phthalate comprising the single-stranded DNA digestor of claim 1.
The phthalate detection composition according to claim 3, wherein the phthalate is diethylhexyl phthalate (DEHP) or monoethylhexyl phthalate (MEHP).
A phthalate detection kit comprising the single stranded DNA extramammary according to claim 1.
The phthalate detection kit according to claim 5, wherein the phthalate is diethylhexyl phthalate (DEHP) or monoethylhexyl phthalate (MEHP).
A phthalate immobilization method on NOS (N-oxysuccinimide) coated plates comprising the steps of:
(a) coating the plate with a NOS group;
(b) immobilizing EDA on the plate surface by reacting EDA (Ethylenediamine) on the coated plate;
(c) sequentially reacting phthalate with EDC (1-Ethyl-3- (3-dimethylaminopropyl) -carbodiimide) and NHS (N-Hydroxysuccinimide);
(d) fixing the phthalate to the NOS coated plate by reacting the reactant of step (c) with a plate on which the EDA is immobilized.
A single stranded DNA extramammary screening method which specifically binds to a phthalate comprising the steps of:
(a) fixing a phthalate to a NOS (N-oxysuccinimide) coated plate by the method of claim 7;
(b) reacting a single-stranded DNA extramamer expected to specifically bind to the phthalate with the phthalate-immobilized NOS-coated plate;
(c) separating the platemer bound to the phthalate.

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