WO2022131846A1 - Method for detecting target material by using graphene oxide and aptamer/g-quadruplex-hexane-structure-based hybridization chain reaction - Google Patents

Abstract

The present invention relates to a method for detecting a target material by forming a G-quadruplex structure through a hybridization chain reaction, which uses an aptamer hairpin probe comprising an aptamer capable of specifically binding to a target material, a first hairpin probe partially hybridizing with the aptamer hairpin probe, and a second hairpin probe partially hybridizing with the first hairpin probe, and using a signal material that specifically recognizes same. Compared with a conventional aptamer-based target material detection method, the present invention enables easier and less expensive detection of a target material under various reaction conditions. In addition, a hairpin probe not involved in the reaction is adsorbed by introducing graphene oxide thereto, and thus a non-specific hybridization chain reaction can be more effectively prevented than a conventional hybridization chain reaction.

Description

Target substance detection method using graphene oxide and aptamer and hybrid chain reaction based on G-QUADRUPLEX nucleic acid construct

The present invention is a target material detection method using graphene oxide and an aptamer and a G-quadruplex nucleic acid structure-based hybridization chain reaction (Graphene oxide and aptamer/G-quadruplex-based hybridization chain reaction, hereinafter also referred to as 'GQ-HCR') In more detail, an aptamer hairpin probe comprising an aptamer capable of specifically binding to a target substance, a first hairpin probe partially hybridizing with the aptamer hairpin probe, and partial hybridization with the first hairpin probe A G-quadruplex nucleic acid construct is formed with a target material using a second hairpin probe to It relates to a method for detecting a target substance using a hybrid chain reaction based.

An aptamer is a single-stranded nucleic acid (DNA or RNA) with a length of about 20 to 60 mers that has a stable secondary structure and can bind to a target material with high affinity and specificity. Aptamers were first developed through an aptamer screening technology called Systematic Evolution of Ligands by EXponential enrichment (SELEX) developed by Larry Gold's research team in 1990. Numerous aptamers that can be combined with Since these aptamers usually have a high binding affinity of pM level, they are spotlighted as substances that can replace antibodies. Unlike antibodies, aptamers are manufactured through chemical synthesis, so they can be produced in a short time and at low cost, and modification is easy. In addition, since it has very high stability to the physical/chemical environment and can be stored and transported for a long time at room temperature, it has the advantage of being very advantageous for field diagnostic applications requiring repeated use for a long time.

Recently, target material detection technology based on the target material-aptamer interaction has been actively studied. Typically, various target material detection technologies using nanoparticle-based aptamers have been developed by using the properties of aptamers that are easy to immobilize on gold or carbon nanoparticles. However, this has a problem in that the detection sensitivity is not good, and an error signal is generated from the instability of the nanoparticles immobilized with the aptamer in the reaction buffer condition (Wang, Y., Li, H., & Xu, D., Analytica chimica). acta, 905, 149-155. (2016); Yi, Y., et al., Analytical Methods, 5 (10), 2477-2484. (2013)). Meanwhile, aptamer-based target material detection technologies using enzymes that induce an amplification reaction for high sensitivity have also been actively developed. However, it is limited in pH, reaction temperature, buffer conditions, etc., and has a disadvantage in that the noise signal is large (Lee, J., et al., Analytical chemistry, 82(1), 197-202.(2010); Yao). , L. Y., et al., Analytical Methods, 7(20), 8786-8792. (2015)).

Meanwhile, there is currently no established method for specifically detecting epithelial cell evidence at the crime scene and using it as evidence to identify the criminal.

Accordingly, the present inventors have made diligent efforts to overcome the above-mentioned limitations of the prior art, and as a result, an aptamer hairpin probe including an aptamer capable of specifically binding to a target material, and a first hairpin partially hybridizing with the hairpin probe When using a probe and a second hairpin probe that partially hybridizes with the first hairpin probe, a G-quadruplex nucleic acid structure is formed together with a target material, and a target material-specific detection signal through amplification of the G-quadruplex nucleic acid structure based on hybrid chain reaction can be effectively amplified, and non-specific reactions can be effectively inhibited by additionally introducing graphene oxide, and epithelial cells can be effectively detected using both the GQ-HCR technology and thioflavin T (ThT). confirmed, and the present invention was completed.

The information described in the background section is only for improving the understanding of the background of the present invention, and it does not include information forming prior art known to those of ordinary skill in the art to which the present invention pertains. it may not be

Summary of the invention

It is an object of the present invention to provide a novel hybrid chain reaction composition and kit capable of detecting a target material with high specificity and sensitivity, and a method for detecting a target material using the same.

Another object of the present invention is to provide a novel composition capable of detecting epithelial cells with high specificity and sensitivity and a method for detecting epithelial cells using the same.

In order to achieve the above object, the present invention provides an aptamer hairpin probe comprising an aptamer and trigger-1 base sequence that specifically binds to a target substance; a first hairpin probe comprising a nucleotide sequence complementary to the trigger-1 nucleotide sequence, a G-rich nucleotide sequence, and a trigger-2 nucleotide sequence; and a second hairpin probe comprising a nucleotide sequence complementary to the trigger-1 nucleotide sequence, the G-rich nucleotide sequence, and the trigger-2 nucleotide sequence.

The present invention also provides a kit for detecting a target material comprising the composition.

The present invention also provides a method for detecting a target substance using a hybrid chain reaction comprising the steps of:

(a) subjecting the target material to a hybrid chain reaction with the composition; and

(b) measuring the G-quadruplex structure formed by the hybrid chain reaction.

The present invention also provides an aptamer hairpin probe comprising an aptamer and trigger-1 base sequence that specifically binds to an epithelial cell protein; a first hairpin probe comprising a nucleotide sequence complementary to the trigger-1 nucleotide sequence, a G-rich nucleotide sequence, and a trigger-2 nucleotide sequence; a second hairpin probe comprising a nucleotide sequence complementary to the trigger-1 nucleotide sequence, the G-rich nucleotide sequence, and the trigger-2 nucleotide sequence; And Thioflavin T (Thioflavin T, ThT), it provides a composition for detecting epithelial cells.

The present invention also provides a method for detecting epithelial cells, comprising reacting a sample containing epithelial cells with the composition for detecting epithelial cells.

1 is a schematic diagram of a method for detecting a target material using the reaction of GQ-HCR technology according to the present invention. Specifically, (A) shows that when the target material is not present, the hybrid chain reaction does not proceed, so that all hairpin probes (HP, H1, H2) are adsorbed to graphene oxide and no fluorescence signal is generated, (B) is a hybrid chain reaction induced by the combination of HP and the target material when a target material is present. H1 and H2 adsorbed on graphene oxide react in a chain reaction to form a G-quadruplex structure in the hybrid chain reaction product. It shows that a strong fluorescence signal is generated by the fluorescence material that binds to the formed G-quadruplex structure.

Figure 2 shows the results of the effectiveness test of the GQ-HCR technology according to an embodiment of the present invention. Fluorescence according to various reaction conditions (a: H1+H2; b: target material + HP + H2; c: target material + HP +H1; d: HP + H1 + H2; e: target material + HP + H1 + H2) Shows the results of an experiment to check whether or not a signal is generated.

3 shows the target material detection sensitivity of the GQ-HCR technique according to an embodiment of the present invention. In (a), the fluorescence intensity according to the wavelength distribution was confirmed using various concentrations of the target material, and in (b) A change in the concentration-dependent fluorescence intensity of the target material was confirmed.

4 shows the target material detection specificity results of the GQ-HCR technique according to an embodiment of the present invention.

5 is a result of confirming the target substance-specific detection effect of the GQ-HCR technique according to an embodiment of the present invention using a target membrane protein of an epithelial cell. SK-BR-3: cells expressing target membrane protein (HER2), MDA-MB-231: cells not expressing target membrane protein (HER2).

6 shows the signal-to-noise ratio (S/N ratio) results according to the presence or absence of graphene oxide of the GQ-HCR technology.

7 is a schematic diagram of a method for detecting target epithelial cells using both GQ-HCR technology and Thioflavin T (ThT) according to the present invention. More specifically, (A) shows that a G-quadruplex structure is formed in the product of a hybrid chain reaction made by a GQ-HCR reaction initiated via a membrane protein expressed on the surface of an epithelial cell, and the formed G-quadruplex structure and ThT It shows that a strong fluorescence signal is generated through the interaction of ThT, and (B) shows that a strong fluorescence signal is generated through the direct interaction between the epithelial cell internal material and ThT.

Figure 8 shows the results of the detection efficacy test of epithelial cells using both GQ-HCR and ThT. Various reaction conditions (a: no target epithelial cells (HaCaT cells); b: no ThT; c: no hairpin probes (HP, H1, H2); d: target membrane protein (HP) among hairpin probes) HER2) shows the experimental results confirming whether a fluorescence signal is generated according to a condition in which a random sequence is replaced; e: a condition in which the target epithelial cell, ThT, and hairpin probe are all included).

9 shows the experimental results for verifying the interaction between the ThT and the epithelial cell internal protein contained in the epithelial cell detection composition. The results of confirming the intensity of the 492 nm fluorescence signal measured in the analysis sample containing various concentrations of the target epithelial cell internal protein (keratin) are shown.

10 shows the detection sensitivity of target epithelial cells using a composition for detecting epithelial cells. In (a), the fluorescence intensity according to the wavelength distribution was confirmed using various concentrations of target epithelial cells, and in (b), target epithelial cells Shows the result of confirming the change in fluorescence intensity at 492 nm according to the concentration of .

11 shows the results of observing epithelial cells of various concentrations visualized using a composition for detecting epithelial cells with a 450 nm method light source and a shielding filter that selectively transmits wavelengths around 492 nm, (a) is the method Epithelial cells before irradiation with light sources are photographed, (b) is images of epithelial cells observed through a shielding filter after irradiation with a method light source, and (c) is a photograph of each reaction solution observed in (a) and (b) This is a table showing the concentration of epithelial cells containing this.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Unless defined otherwise, 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 those well known and commonly used in the art.

In one embodiment of the present invention, graphene oxide and aptamer and G-quadruplex nucleic acid structure-based hybridization chain reaction (Graphene oxide and aptamer/G-quadruplex-based hybridization chain reaction, GQ-HCR) technology to effectively target material through It was confirmed that it could be detected. Specifically, in the present invention, an aptamer hairpin probe comprising an aptamer capable of specifically binding to a target material, a first hairpin probe partially hybridized with the aptamer hairpin probe, and a second hairpin probe partially hybridized with the first hairpin probe Using a hairpin probe, a G-quadruplex nucleic acid structure was formed with a target material, and a target material-specific detection signal was effectively amplified through hybrid chain reaction-based G-quadruplex nucleic acid structure amplification, and non-specific graphene oxide was introduced. The reaction was effectively inhibited.

Accordingly, the present invention provides an aptamer hairpin probe comprising an aptamer and trigger-1 nucleotide sequence that specifically binds to a target material in one aspect; a first hairpin probe comprising a nucleotide sequence complementary to the trigger-1 nucleotide sequence, a G-rich nucleotide sequence, and a trigger-2 nucleotide sequence; and a second hairpin probe comprising a nucleotide sequence complementary to the trigger-1 nucleotide sequence, the G-rich nucleotide sequence, and the trigger-2 nucleotide sequence.

In another aspect, the present invention relates to a kit for detecting a target material comprising the composition.

In the present invention, the composition may be characterized in that it further comprises graphene oxide. This is to suppress the non-specific amplification reaction by introducing graphene oxide having the property of adsorbing single-stranded nucleic acids so that the hairpin probe used for GQ-HCR can be exposed to the reaction solution only when it participates in the reaction.

In the present invention, the composition may be characterized in that it further comprises PBS, NaCl, MgCl2.

In the present invention, the aptamer hairpin probe may also be denoted as 'HP', and may include an aptamer and a trigger-1 base sequence, but may further include a space sequence between the aptamer and the trigger-1 base sequence. As shown in FIG. 1 , in the aptamer hairpin probe, a part of the aptamer base sequence and a part of the trigger-1 base sequence are hybridized to form a hairpin structure.

In the present invention, the first hairpin probe includes a nucleotide sequence complementary to the trigger-1 nucleotide sequence, a G-rich nucleotide sequence, and a trigger-2 nucleotide sequence. In this case, as shown in FIG. 1 , in the first hairpin probe, a part of a nucleotide sequence complementary to the trigger-1 nucleotide sequence and a part of the trigger-2 nucleotide sequence are hybridized to form a hairpin structure.

In the present invention, the second hairpin probe includes a nucleotide sequence complementary to the trigger-1 nucleotide sequence, the G-rich nucleotide sequence, and the trigger-2 nucleotide sequence. In this case, as shown in FIG. 2 , in the second hairpin probe, a part of the nucleotide sequence complementary to the trigger-2 and a part of the trigger-1 nucleotide sequence are hybridized to form a hairpin structure.

In the present invention, the trigger-1 nucleotide sequence may also be expressed as 'hybrid chain reaction inducing nucleotide sequence-1', and the trigger-2 nucleotide sequence may also be expressed as 'hybrid chain reaction inducing nucleotide sequence-2' . In the present invention, the trigger-1 nucleotide sequence is exposed by binding the aptamer hairpin probe to the target material, the first hairpin probe binding to the exposed trigger-1 nucleotide sequence, and the trigger-2 nucleotide sequence exposed and exposed The second hairpin probe is bound to the trigger-2 base sequence, the trigger-1 base sequence of the bound second hairpin probe is exposed, and another first hairpin probe is bound thereto and the trigger-2 base sequence is exposed. Likewise, a hybrid chain reaction is induced by these trigger sequences.

In the present invention, when the target material is not present, three types of hairpin probes (HP, H1, H2) are adsorbed to graphene oxide and do not participate in the reaction. However, when a target material is present, the aptamer hairpin probe adsorbed on graphene oxide is exposed to the reaction solution by binding to the target material. At this time, the trigger-1 base sequence is exposed as the aptamer hairpin probe binds to the target material and the structure is modified. The first hairpin probe is opened by the strand displacement reaction to expose the G-rich base sequence and the trigger-2 base sequence. The exposed trigger-2 base sequence binds to the scaffold of the second hairpin probe, and the second hairpin probe is opened by a strand displacement reaction to expose the G-rich base sequence and the trigger-1 base sequence. In this reaction, the trigger-2 base sequence inserted into the first hairpin probe and the trigger-1 base sequence inserted into the first hairpin probe are continuously exposed, and the first hairpin probe and the second hairpin probe are sequentially opened. As a result, a double-stranded nucleic acid, a hybrid chain reaction product, is generated and a large amount of G-rich nucleotide sequences are freely exposed. The free G-rich nucleotide sequence forms a G-quadruplex structure, and by introducing a fluorescent substance (Thioflavin T, ThT) that specifically binds to G-quadruplex and generates a strong fluorescence signal, the strong fluorescence signal amplified from ThT can be checked

Accordingly, the composition may further include a signal material that specifically binds to a product produced by the reaction of the aptamer hairpin probe, the first hairpin probe, and the second hairpin probe, wherein the produced product may be characterized as a G-quadruplex structure.

In the present invention, the signal material includes any one or more selected from the group consisting of a radiolabel, a fluorophore, a chromophore, an imaging agent, and a metal ion. It may be characterized in that, but is not limited thereto.

In the present invention, preferably, the signal material may be Thioflavin T (Thioflavin T, ThT). Thioflavin T binds specifically to the G-quadruplex structure generated by the hybrid chain reaction and amplifies the fluorescence signal, thereby realizing a non-enzyme-free and label-free high-sensitivity detection system. have. In this case, if it is a signal material that specifically binds to the G-quadruplex structure and shows a strong fluorescence signal, it will be possible to add another fluorescent material that can replace Thioflavin T.

That is, in the present invention, the probe is modified with a separate signal material, and can generate a signal by itself by performing a hybrid chain reaction in the presence of a target material. - It may be characterized in that it forms a quadruplex nucleic acid construct, and a signal material specifically binds thereto.

In the present invention, the G-rich nucleotide sequence is preferably composed of 3 to 50, preferably 5 to 30, more preferably 7 to 15, and most preferably 12 guanines (G), but limited thereto it doesn't happen

In the present invention, the target material may be selected from the group consisting of proteins, peptides, metal ions, low molecular weight organic substances and small molecules, but is not limited thereto.

In the present invention, the protein or peptide may be a disease-specific protein or peptide, and in this case, the present invention may be utilized for disease diagnosis.

In the present invention, the protein or peptide may be a cell membrane protein, for example, EpCAM or HER2, for example, may be a cell membrane protein expressed so that a part is exposed on the cell surface, in this case, the protein without cell disruption However, it has the advantage of being able to detect peptides.

In an embodiment of the present invention, a hairpin probe (aptamer hairpin, HP) in which a hybrid chain reaction inducing nucleotide sequence-1 (trigger-1) and an aptamer nucleotide sequence specific to a target material are modified, adsorbed to graphene oxide ) is exposed to the reaction solution by binding to the target substance and the structure of HP is modified to expose the trigger-1 base sequence; A hairpin in which a nucleotide sequence complementary to Trigger-1 adsorbed to graphene oxide, a G-rich nucleotide sequence, and a hybrid chain reaction inducing nucleotide sequence-2 (Trigger-2) is modified by the exposed trigger-1 nucleotide sequence A hairpin probe (HCR hairpin-2, hereinafter H2) modified with a probe (HCR hairpin-1, hereinafter H1) and a trigger-1 sequence, a G-rich nucleotide sequence, and a nucleotide sequence complementary to a trigger-2 reaction solution is exposed to a hybridization reaction; The product generated by the hybrid chain reaction formed a G-quadruplex structure, and a fluorescent substance that specifically binds to the G-quadruplex structure and shows a strong fluorescence signal was added to confirm the fluorescence signal.

Accordingly, the present invention relates to a method for detecting a target substance using a hybrid chain reaction comprising the following steps from another aspect:

(a) subjecting the target material to a hybrid chain reaction with the composition; and

(b) measuring the G-quadruplex structure formed by the hybrid chain reaction.

In the present invention, the step (a) is

(i) exposing the trigger-1 sequence of the aptamer hairpin probe by reacting the aptamer hairpin probe with a target material; and (ii) inducing a hybridization chain reaction by opening the first hairpin probe and the second hairpin probe in a chain by the trigger-1 nucleotide sequence exposed in step (i). It may be characterized in that (FIG. 1).

In the present invention, the hybrid chain reaction is

i) exposing the trigger-2 base sequence by binding the trigger-1 base sequence of the aptamer hairpin probe to a base sequence complementary to the trigger-1 base sequence of the first hairpin probe;

ii) exposing the trigger-1 base sequence by binding the exposed trigger-2 base sequence to a base sequence complementary to the trigger-2 base sequence of the second hairpin probe; and

iii) the exposed trigger-1 base sequence binds to a base sequence complementary to the trigger-1 base sequence of the first hairpin probe to expose the trigger-2 base sequence, and steps ii) and iii) are repeated. By doing so, it may be characterized in that the first hairpin probe and the second hairpin probe are opened in series.

In the present invention, the opening of the first hairpin probe means that the trigger-2 sequence and the G-rich sequence of the first hairpin probe are exposed, and the opening of the second hairpin probe means that the second hairpin probe is opened. It means that the trigger-1 nucleotide sequence and the G-rich nucleotide sequence are exposed.

The GQ-HCR technology according to the present invention induces a hybrid chain reaction, an isothermal, non-enzyme-free signal amplification reaction, using an aptamer hairpin probe containing an aptamer sequence specific to a target material. To describe this in more detail, when the target material is present, the hairpin structure of the aptamer hairpin probe containing the aptamer sequence is released and linearized by the target material, and another two types of the linearized aptamer hairpin probe The hairpin probe of the reaction was designed to operate the hybrid chain reaction. In this case, since a large amount of guanine base sequence (hereinafter referred to as G-rich base sequence) is inserted in the two additional haypin probes, the hybrid chain reaction product can be effectively formed into a G-quadruplex structure. On the other hand, in the present invention, a fluorescent material that specifically binds to the G-quadruplex structure is introduced, and the hybrid chain reaction is amplified by binding aptamer hairpin graphene to the target material and additionally binding two types of hairpin probes. When the fluorescent material binds to the G-quadruplex structure, a strong fluorescent signal is generated, and the target material is detected by measuring the fluorescent signal.

On the other hand, it is preferable that the reaction proceeds in a reaction composition containing graphene oxide. In this case, in the absence of the target material, three types of hairpins (HP, H1, H2) are adsorbed to the graphene oxide and do not participate in the reaction, whereas when the target material is present, they are adsorbed to the graphene oxide. The present HP is exposed to the reaction solution by binding to the target material.

In the present invention, the target material may be characterized in that it contains any one or more selected from the group consisting of disease-derived proteins, cell membrane proteins, small molecules, and metal ions, and the cell membrane proteins are preferably EpCAM and HER2. may be, but is not limited thereto.

In the present invention, when the target material is not present, the aptamer hairpin probe, the first hairpin probe, and the second hairpin probe may be characterized in that they are adsorbed to graphene oxide.

In the present invention, the aptamer hairpin probe may include a trigger-1 base sequence and an aptamer binding to a target material.

In the present invention, the first hairpin probe may include a nucleotide sequence complementary to a trigger-1 nucleotide sequence, a G-rich nucleotide sequence, and a trigger-2 nucleotide sequence.

In the present invention, the second hairpin probe may include a nucleotide sequence complementary to a trigger-2 nucleotide sequence, a G-rich nucleotide sequence, and a trigger-1 nucleotide sequence.

In the present invention, the G-quadruplex structure of step (b) may be characterized in that it comprises a G-rich nucleotide sequence.

In the present invention, the G-quadruplex structure of step (b) is detected as a signal material, and the signal material is a radiolabel, a fluorophore, a chromophore, an imaging agent and It may be characterized in that it contains any one or more selected from the group consisting of metal ions, and preferably, the signal material may be Thioflavin T (ThT), but is limited thereto not.

In the present invention, the aptamer hairpin probe, the first hairpin probe, and the second hairpin probe of a specific nucleotide sequence were used, but when using a combination of nucleotide sequences capable of hybrid chain reaction as shown in FIG. 1 of the present invention, the present invention It will be apparent to those skilled in the art that the invention can be implemented in various ways.

On the other hand, it is not easy to specifically detect oiled epithelial cells in order to use them as evidence to identify criminals at the crime scene, and in one embodiment of the present invention, three types of hairpin probes (HP , H1, H2), a very low fluorescence signal is generated (condition b), so there is a limit to detecting epithelial cells, whereas all three hairpin probes (HP, H1, H2) and ThT are used. When the composition containing the composition was used, a stronger fluorescence signal was generated (condition e), so it was confirmed that epithelial cells could be effectively detected.

Accordingly, in the present invention, (a) a fluorescence signal generated due to the interaction of ThT with G-quadruplex formed by the GQ-HCR reaction mediated by the membrane protein expressed on the epithelial cell surface and (b) ThT and the inside of the epithelial cell Epithelial cells were detected by measuring all the fluorescence signals generated due to the interaction with the substance (Example 8, FIGS. 7 and 8).

Accordingly, in another aspect, the present invention provides an aptamer hairpin probe comprising an aptamer and trigger-1 base sequence that specifically binds to an epithelial cell protein; a first hairpin probe comprising a nucleotide sequence complementary to the trigger-1 nucleotide sequence, a G-rich nucleotide sequence, and a trigger-2 nucleotide sequence; a second hairpin probe comprising a nucleotide sequence complementary to the trigger-1 nucleotide sequence, the G-rich nucleotide sequence, and the trigger-2 nucleotide sequence; And Thioflavin T (Thioflavin T, ThT), it relates to a composition for detecting epithelial cells.

In the present invention, the epithelial cell protein is characterized in that it is an epithelial cell membrane protein, preferably EpCAM or HER2, but is not limited thereto.

In the present invention, the thioflavin T may be characterized in that it generates a signal by reacting with the material inside the epithelial cells.

Thioflavin T (ThT) used in the present invention is not only a substance capable of generating a fluorescence signal through interaction with G-quadruplex, but also interaction with internal substances such as keratin present in epithelial cells. It is a material that can generate a fluorescence signal through

In the present invention, the epithelial cell internal material includes all substances that generate a fluorescence signal by binding to ThT, and the term “inside” refers to the cytoplasm, the nucleus, and other organelles present in the cell membrane. The epithelial cell internal material is preferably an internal protein, more preferably keratin, but is not limited thereto.

In another aspect, the present invention relates to a method for detecting epithelial cells, comprising reacting a sample containing epithelial cells with the composition for detecting epithelial cells.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example

Example 1. Establishment of reaction conditions for GQ-HCR technology

The process of preparing the reaction solution of the GQ-HCR technology according to the present invention is as follows.

In a reaction buffer containing 10 mM PBS (pH 6.0), 100 mM NaCl, and 10 mM MgCl2, 0.9 µL of HP (1 µM), 1.2 µL of H1 (10 µM), 1.5 µL of H2 (10 µM), and graphene oxide ( 1 mg/mL) and 6 µL of target substances of various concentrations are added to the reaction buffer solution to prepare a reaction solution (30 µL final), and the reaction is allowed to proceed at 37°C for 1 hour. After the reaction was completed, 1.5 uL of ThT (2.5 mM) was added to the reaction solution, and the final G-quadruplex structure production amount was analyzed by measuring the intensity of a fluorescence signal generated from ThT.

The nucleotide sequence information of the nucleic acid probe used for the development of this GQ-HCR technology is as follows, but is not limited thereto.

HP: 5'-GTA GCC TGA AGC CAA ACC AAA AAA AAA AAC AGG CTA CGG CAC GTA GAG CAT CAC CAT GAT CCT G-3' (SEQ ID NO: 1)

H1: 5'-TGG TTT GGC TTC AGG CTA CGG CTG GGT AGG GCG GGT TGG GAA AAA TCC AGC CGT AGC CTG-3' (SEQ ID NO: 2)

H2: 5'- GCC GTA GCC TGA AGC CAA ACC ATG GGT AGG GCG GGT TGG GAA ACA GGC TAC GGC TGG ATT-5' (SEQ ID NO: 3)

Example 2. Validation of GQ-HCR Technology

A validation experiment of the present technology was conducted using the reaction conditions mentioned in Example 1. To this end, a validation experiment was conducted using Platelet-derived growth factor BB (hereinafter referred to as PDGF-BB) as a target material. As a result of performing experiments according to various reaction conditions (a to e), it was confirmed that an exceptionally high fluorescence signal was generated under the reaction conditions (e) including all of the target substances, PDGF-BB, HP, H1, and H2 (Fig. 2).

Example 3. Verification of target substance detection sensitivity of GQ-HCR technology

A sensitivity verification experiment of the present technology was performed using the reaction conditions mentioned in Example 1. As a result of preparing an assay sample containing various concentrations (50 pM to 20 nM) of the target material (PDGF-BB) and performing the assay reaction, the limit of detection (LOD) of the present technology is 4.53 pM It was confirmed that this was an excellent level of performance comparable to existing technologies (FIG. 3).

Example 4. Verification of target substance detection specificity of GQ-HCR technology

The selectivity verification experiment of the present technology was conducted using the reaction conditions mentioned in Example 1. An assay reaction was performed on an assay sample (30 μL) containing 400 nM of non-target substances (Thrombin, BSA, Cytochrome C, Lysozyme, Immunoglobulin G) and 20 nM of PDGF-BB. A strong fluorescence signal was generated only in the analyte sample, and accordingly, it was confirmed that the target material, PDGF-BB, could be selectively detected based on the present technology (FIG. 4).

Example 5. Validation of Target Membrane Protein Detection Using GQ-HCR Technology

Using the reaction conditions mentioned in Example 1, a validation experiment for the detection of the target membrane protein of the present technology was conducted using epithelial cells overexpressing the target membrane protein. Analysis sample containing 2,500 cells/μL of target membrane protein (HER2) overexpressing epithelial cells, SK-BR-3, and 2,500 cells/μL of target membrane protein (HER2) non-expressing epithelial cells, MDA-MB-231 As a result of performing the analysis reaction after preparing the analysis sample and the analysis sample that does not contain epithelial cells, an exceptionally high fluorescence signal is generated only under the reaction conditions containing the target membrane protein (HER2) overexpressing epithelial cell, SK-BR-3. was confirmed (FIG. 5).

Example 6. Comparison of S/N ratio with or without graphene oxide

The validation experiment of the present invention was conducted using the reaction conditions mentioned in Example 1. As the target material, 20 nM of PDGF-BB used in Example 2 was used, and the experiment was conducted under the same conditions except for the presence or absence of graphene oxide. Signal-to-ratio (S/N Ratio) is the fluorescence signal value (F) of the positive sample containing the target substance compared to the fluorescence signal value of the negative sample that does not contain the target substance, which is commonly used between studies. (F0) was calculated by applying the ratio (F/F0), and as a result, when graphene oxide was introduced as shown in FIG. 6, the signal-to-noise ratio was significantly high, and the introduction of graphene oxide according to the present invention It was confirmed that the non-specific reaction of GQ-HCR technology can be effectively suppressed.

Example 7. Construction of a composition for detecting epithelial cells using both GQ-HCR technology and thioflavin T (ThT)

The preparation process of the reaction solution for epithelial cell detection (hereinafter, epithelial cell detection composition) using both GQ-HCR technology and ThT according to the present invention is as follows.

In a reaction buffer containing 10 mM PBS (pH 6.0), 100 mM NaCl, and 10 mM MgCl2, 0.9 µL of HP (1 µM), 1.2 µL of H1 (10 µM), 1.5 µL of H2 (10 µM), and graphene oxide ( 1 mg/mL) and 6 µL of target epithelial cells of various concentrations are added to the reaction buffer to prepare a reaction solution (final 30 µL), and the reaction is allowed to proceed at 37 °C for 5 min. After the reaction was completed, 3.6 uL of ThT (2.5 mM) was added to the reaction solution, and the intensity of a fluorescence signal generated from ThT was measured.

Base sequence information of the nucleic acid probe used for the detection of epithelial cells using the composition for detecting epithelial cells is as follows, but is not limited thereto.

HP: 5'-CGC TGC AAG CCA AAC CAA AAA AAA AAA GCA GCG GTG TGG GGG CAG CGG TGT GGG GGC AGC GGT GTG GGG-3' (SEQ ID NO: 4)

H1: 5'-TGG TTT GGC TTG CAG CGG GCT GGG TAG GGC GGG TTG GGA AAA ATC CAG CCC GCT GCA A-3' (SEQ ID NO: 5)

H2: 5'-GCC CGC TGC AAG CCA AAC CAT GGG TAG GGC GGG TTG GGA AAT TGC AGC GGG CTG GAT T-3' (SEQ ID NO: 6)

Example 8. Evaluation of Efficacy of Detection of Epithelial Cells Using Both GQ-HCR and ThT

An epithelial cell detection validation experiment using the epithelial cell detection composition was performed using the reaction conditions mentioned in Example 7 (FIG. 8). As a result of performing experiments according to various reaction conditions (a to e), a low fluorescence signal was generated in the sample in which the target membrane protein or ThT was not present in the reaction solution (condition a, b), but both epithelial cells and ThT were In the case of the included sample, it was confirmed that a strong fluorescence signal was generated (conditions c ~ e). In particular, even when hybridization chain reaction (HCR) by the target membrane protein (HER2) does not occur (condition c, d), when HCR occurs when the target epithelial cell (HaCaT cell) is present (condition e), about 80% By confirming that the fluorescence signal of It was confirmed that a stronger fluorescence signal was generated when (Fig. 8).

Example 9. Verification of the interaction between ThT and epithelial cell internal protein using a composition for detecting epithelial cells

An experiment was conducted to verify the interaction between the ThT contained in the GQ-HCR reaction solution and the epithelial cell internal protein using a composition for detecting epithelial cells. To this end, the fluorescence signal generated due to the interaction between keratin, which accounts for most of the inner protein of epithelial cells, and ThT contained in the composition for detecting epithelial cells was measured. After preparing an assay sample by adding various concentrations of keratin (11.05 ng/μL ~ 221 ng/μL) to the composition for detecting epithelial cells instead of epithelial cells, the assay reaction was performed. As a result, the fluorescence signal generated as the concentration of keratin increased By confirming that , it was confirmed that keratin inside epithelial cells reacted with ThT contained in the composition for detecting epithelial cells to generate a strong fluorescence signal (FIG. 9).

Example 10. Detection of target epithelial cells using a composition for detecting epithelial cells and confirmation of detection limits

An experiment to verify the detection sensitivity of target epithelial cells was performed using the composition for detecting epithelial cells. After preparing an assay sample containing various concentrations (10 cells/μL ~ 10,000 cells/μL) of target epithelial cells (HaCaT cells), the assay reaction was performed. As a result, the target epithelial cell detection limit of the present technology LOD) was confirmed to be 4.10 cells/μL (FIG. 10).

Example 11. Visualization result of epithelial cells using beop light source

An experiment was conducted to determine whether target epithelial cells could be observed using a forensic light source used in a crime scene through a reaction using a composition for detecting epithelial cells. A composition for detecting epithelial cells was applied on a Petri dish coated with various concentrations (10 cells/μL ~ 10,000 cells/μL) of target epithelial cells (HaCaT cells) to proceed with the reaction, and then the ThT excitation wavelength using a method light source Signals generated in target epithelial cells of various concentrations were observed using a shielding filter that selectively transmits a wavelength of 450 nm corresponding to 450 nm and selectively transmits a wavelength around 492 nm. As a result, it was confirmed that the intensity of the observed fluorescence signal became stronger as the concentration of epithelial cells increased. was confirmed (FIG. 11).

The target material detection method according to the present invention is more stable than the conventional aptamer-based target material detection method using nanoparticles and enzymes, and has the advantage of being able to detect the target material more cheaply and conveniently without restrictions on various reaction conditions. In addition, the target material detection method according to the present invention does not require immobilization of a fluorescent material-modified nucleic acid probe, fluorescent material labeling, or an enzyme amplifying nucleic acid, and is non-enzyme-free and label-free. ) can be detected. In addition, the present invention is significant in that non-specific reactions can be dramatically prevented by adsorbing hairpin probes that do not participate in the reaction using graphene oxide. Therefore, the target material detection method according to the present invention can target all substances detectable with an aptamer, such as disease-derived proteins, cell membrane proteins such as epithelial cells, small molecules, and metal ions, and can be used in various fields. It is meaningful in that it is a technology that can do this and has excellent versatility.

In addition, when the composition for detecting epithelial cells according to the present invention is used together with a forensic light source at a crime scene, it can be utilized for visualization of epithelial cell evidence and further contribute to the development of forensic investigation.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

An electronic file is attached.

Claims (24)

1. an aptamer hairpin probe comprising an aptamer and trigger-1 base sequence that specifically binds to a target substance;

   a first hairpin probe comprising a nucleotide sequence complementary to the trigger-1 nucleotide sequence, a G-rich nucleotide sequence, and a trigger-2 nucleotide sequence; and

   A composition for detecting a target material, comprising a second hairpin probe comprising a nucleotide sequence complementary to the trigger-1 nucleotide sequence, the G-rich nucleotide sequence, and the trigger-2 nucleotide sequence.
2. The composition of claim 1, wherein the composition further comprises graphene oxide.
3. According to claim 1, wherein the composition further comprises a signal material that specifically binds to a product produced by the reaction of the aptamer hairpin probe, the first hairpin probe, and the second hairpin probe, target substance detection for composition.
4. The composition for detecting a target material according to claim 3, wherein the generated product is a G-quadruplex structure.
5. The composition for detecting a target material according to claim 4, wherein the G-quadruplex structure comprises a G-rich nucleotide sequence.
6. The method of claim 3, wherein the signal material is any one or more selected from the group consisting of a radiolabel, a fluorophore, a chromophore, an imaging agent, and a metal ion. A composition for detecting a target material, comprising:
7. The composition for detecting a target material according to claim 3, wherein the signal material is Thioflavin T (ThT).
8. The composition of claim 1, wherein the target material comprises at least one selected from the group consisting of disease-derived proteins, cell membrane proteins, small molecules, and metal ions.
9. A kit for detecting a target material comprising the composition of any one of claims 1 to 8.
10. A method for detecting a target substance using a hybrid chain reaction comprising the following steps:

    (a) subjecting the target material to a hybrid chain reaction with the composition of any one of claims 1 to 8; and

    (b) measuring the G-quadruplex structure formed by the hybrid chain reaction.
11. 11. The method of claim 10, wherein (a) step

    (i) exposing the trigger-1 sequence of the aptamer hairpin probe by reacting the aptamer hairpin probe with a target material; and

    (ii) by the trigger-1 base sequence exposed in step (i), the first hairpin probe and the second hairpin probe are chained open to induce a hybridization chain reaction How to characterize.
12. The method according to claim 10, wherein the target material comprises any one or more selected from the group consisting of disease-derived proteins, cell membrane proteins, small molecules, and metal ions.
13. The method of claim 11, wherein when the target material is not present, the aptamer hairpin probe, the first hairpin probe, and the second hairpin probe are adsorbed to graphene oxide.
14. The method of claim 11, wherein the aptamer hairpin probe comprises a trigger-1 base sequence and an aptamer binding to a target material.
15. The method of claim 11, wherein the first hairpin probe comprises a nucleotide sequence complementary to a trigger-1 nucleotide sequence, a G-rich nucleotide sequence, and a trigger-2 nucleotide sequence.
16. 12. The method of claim 11, wherein the second hairpin probe comprises a nucleotide sequence complementary to a trigger-2 nucleotide sequence, a G-rich nucleotide sequence, and a trigger-1 nucleotide sequence.
17. The method of claim 10, wherein the G-quadruplex structure of step (b) comprises a G-rich nucleotide sequence.
18. The method according to claim 10, wherein the G-quadruplex structure of step (b) is detected as a signal material, and the signal material is a radiolabel, a fluorophore, a chromophore, and an imaging agent. And a method comprising any one or more selected from the group consisting of metal ions (metal ions).
19. The method of claim 18, wherein the signal material is Thioflavin T (ThT).
20. an aptamer hairpin probe comprising an aptamer and trigger-1 base sequence that specifically binds to an epithelial cell protein;

    a first hairpin probe comprising a nucleotide sequence complementary to the trigger-1 nucleotide sequence, a G-rich nucleotide sequence, and a trigger-2 nucleotide sequence;

    a second hairpin probe comprising a nucleotide sequence complementary to the trigger-1 nucleotide sequence, the G-rich nucleotide sequence, and the trigger-2 nucleotide sequence; and

    Thioflavin T (Thioflavin T, ThT), comprising a composition for detecting epithelial cells.
21. The composition of claim 20, wherein the epithelial cell protein is an epithelial cell membrane protein.
22. The composition of claim 21, wherein the epithelial cell membrane protein is EpCAM or HER2.
23. The composition according to claim 20, wherein the Thioflavin T reacts with an epithelial cell internal material to generate a signal.
24. A method for detecting epithelial cells comprising the step of reacting an epithelial cell-containing sample with the composition of any one of claims 20 to 23.

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