Method for detecting hydrogen peroxide by utilizing self-biotinylation of guanine quadruplex

Abstract

The present invention relates to a composition, a kit and a detection method for detecting hydrogen peroxide on the basis of self-biotinylation according to the peroxidation activity of a guanine quadruplex, and, more specifically, to a composition, a kit and a detection method for detecting hydrogen peroxide, the composition comprising: i) a guanine quadruplex-hemin complex (G-quadruplex-hemin complex); ii) biotinyl tyramide; and iii) a probe capable of binding to the guanine quadruplex.

Description

Hydrogen peroxide detection method using autobiotinylation of guanine tetramers

[0001] The present invention relates to a composition, kit, and detection method for detecting hydrogen peroxide based on self-biotinylation according to the peroxidation activity of a guanine quadruplex, and more specifically, to a composition, kit, and detection method for detecting hydrogen peroxide comprising i) a guanine quadruplex-hemin complex; ii) biotinyl tyramide; and iii) a probe capable of binding to the guanine quadruplex.

[0002]

[0003] Hydrogen peroxide (H2O2) is a key biomolecule that regulates intracellular signaling pathways involved in cell proliferation, differentiation, and apoptosis. It is a non-radical byproduct of cellular oxidative metabolism. Furthermore, H2O2 acts as a potent antimicrobial agent, used to combat pathogens that invade the immune system. Therefore, H2O2 dysregulation is closely linked to various oxidative stress-related diseases, including neurodegenerative diseases, cardiovascular diseases, cancer, and inflammatory diseases. Therefore, accurate detection and quantitative analysis of H2O2 are crucial.

[0004] Among the existing known H2O2 detection methods, the most widely used method is the method using horseradish peroxidase (HRP). Specifically, this method analyzes H2O2 in a sample by measuring specific signals such as fluorescence, luminescence, and color that are generated by HRP oxidizing signal substances using H2O2. However, the analysis process of most techniques is cumbersome and time-consuming, and expensive optical analysis equipment is required to measure and analyze the generated signals. [Mohanty et al., J Immunol Methods, 202(2), 133-141.(1997); Summers et al., Method Enzymol, 526, 1-17.(2013); Wang et al., Analytica Chimica Acta, 963, 61-67.(2017); Wymann et al., Anal Biochem, 165(2), 371-378.(1987); Virel et al., Analyst, 135(9), 2291-2295.(2010)] Therefore, there is a need to develop a user-friendly H2O2 detection technology that can solve the price problem of existing equipment and can be easily used by the general public.

[0005] To this end, H2O2 detection methods utilizing strip-based lateral flow assay (LFA), one of the field diagnostic technologies, have been recently developed. [Fung et al., Anal Chim Acta, 634(1), 89-95.(2009); Ozalp et al., Analyst, 138(15), 4255-4259.(2013)] LFA has the advantages of being easy to carry and having a short analysis time, but the existing H2O2 detection technologies have limitations such as requiring complex procedures such as direct immobilization of HRP on the strip or synthesis of nanoparticles containing HRP, or low analytical reliability due to the absence of a control line.

[0006] Under these backgrounds, the present inventors have made efforts to develop a method capable of detecting H2O2 with high sensitivity while being performed with a simple procedure. As a result, by utilizing the fact that self-biotinylation of G-quadruplex occurs when biotinyl tyramide is used as a substrate by the peroxidase activity of G-quadruplex, detection probes, etc. were designed so that this can be applied to the lateral flow spectrometry method. As a result, it was confirmed that the biotin-labeled G-quadruplex formed in the presence of hydrogen peroxide was detected as a color signal in an LFA strip, and the present invention was completed to enable the simple detection and quantification of H2O2.

[0007]

[0008] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0009]

[0010] Summary of the invention

[0011] The purpose of the present invention is to provide a composition, use, kit, and method for detecting hydrogen peroxide that can detect hydrogen peroxide simply and with high reliability by utilizing the self-biotinylation reaction of G-quadruplex.

[0012] Another object of the present invention is to provide a method for detecting a biomolecule capable of generating hydrogen peroxide by an oxidation reaction using the composition and kit.

[0013]

[0014] To achieve the above object, the present invention provides a composition for detecting hydrogen peroxide, comprising i) a guanine quadruplex-hemin complex; ii) biotinyl tyramide; and iii) a probe capable of binding to the guanine-quadruplex.

[0015] The present invention also provides a use for detecting hydrogen peroxide of i) a guanine quadruplex-hemin complex; ii) biotinyl tyramide; and iii) a probe capable of binding to the guanine-quadruplex.

[0016] The present invention also provides a kit comprising the composition.

[0017] The present invention also provides a method for detecting hydrogen peroxide, comprising the steps of: (a) adding the composition to a sample containing hydrogen peroxide and reacting the same; and (b) detecting hydrogen peroxide by detecting a binding product of a self-biotinylated guanine quadruplex and a probe.

[0018] The present invention provides a method for detecting a biomolecule capable of generating hydrogen peroxide by an oxidation reaction, comprising the steps of: (a) adding an oxidizing enzyme to a sample containing a biomolecule capable of generating hydrogen peroxide by an oxidation reaction and reacting the sample; (b) adding the composition to the sample and reacting the sample; and (c) detecting a binding product of a self-biotinylated guanine quadruplex and a probe to detect the biomolecule capable of generating hydrogen peroxide by an oxidation reaction.

[0019]

[0020] Figure 1 is a schematic diagram of the SelBio-LFA technology according to the present invention, which shows a method for detecting H2O2 in a detection sample using a lateral flow immunoassay technique based on the self-biotinylation reaction of G-quadruplex.

[0021] Figure 2 shows the results of an experiment to prove the effectiveness of the SelBio-LFA technology according to the present invention.

[0022] Figure 3 shows the results of an H2O2 detection sensitivity experiment of the SelBio-LFA technology according to the present invention.

[0023] Figure 4 shows the results of an H2O2 detection specificity experiment of the SelBio-LFA technology according to the present invention.

[0024] FIG. 5 schematically illustrates a method for detecting choline, a model biomolecule, using SelBio-LFA technology to demonstrate the possibility of using the present invention for detecting other biomolecules, and shows the results of a choline detection sensitivity experiment.

[0025]

[0026] Detailed description of the invention and preferred embodiments

[0027] 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 pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0028]

[0029] The hydrogen peroxide detection technology utilizing the self-biotinylation reaction of guanine quadruplex provided by the present invention enables reliable detection in a simple manner compared to conventional hydrogen peroxide detection technologies. Conventional hydrogen peroxide detection technologies utilize HRP and have the problem of requiring expensive optical analysis equipment for detection or the manufacturing process of nanoparticles and strips attached to HRP, making manufacturing and detection complicated. The hydrogen peroxide detection technology according to the present invention solves the technical problems of conventional detection technologies by utilizing the self-biotinylation of guanine quadruplex in the presence of hydrogen peroxide.

[0030] In one embodiment of the present invention, it was confirmed that hydrogen peroxide can be detected with high reliability in a sample by utilizing a composition comprising i) a guanine quadruplex-hemin complex; ii) biotinyl tyramide; and iii) a probe capable of binding to the guanine quadruplex (Example 2, FIG. 4).

[0031] It has been reported that the guanine quadruplex-hemin complex has peroxidase activity, catalyzing the peroxidation reaction using hydrogen peroxide and biotinylated tyramide as substrates, and as a result, the guanine quadruplex can be self-biotinylated (Owen J. Einarson, et al., Nucleic Acids Research, Volume 45, Issue 17, 29 September 2017, Pages 9813-9822).

[0032] In a specific embodiment, i) when hydrogen peroxide is present in a sample, a peroxidase activity of a guanine quadruplex-hemin complex induces a peroxidation reaction, thereby causing the guanine quadruplex to be self-biotinylated, and the self-biotinylated guanine quadruplex and the probe to bind to generate a binding product in which both biotin and a label are bound, and the binding product binds to a ligand that specifically binds to biotin, and ii) when hydrogen peroxide is not present in the sample, the guanine quadruplex is not self-biotinylated, and the guanine quadruplex and the probe to generate a binding product in which biotin is not bound, and the hybridization product in which biotin is not bound does not bind to a ligand that specifically binds to biotin, so that it was confirmed that hydrogen peroxide can be indirectly detected with high sensitivity by detecting the binding product in which biotin is bound.

[0033]

[0034] Composition for detecting hydrogen peroxide

[0035] Accordingly, the present invention relates, in one aspect, to a composition for detecting hydrogen peroxide, comprising:

[0036] i) G-quadruplex-hemin complex;

[0037] ii) biotinyl tyramide; and

[0038] iii) A probe capable of binding to the above guanine-quadruplex.

[0039] The term "G-quadruplex" of the invention refers to a structure formed by vertically stacking two or more G-tetraplexes that appear in a DNA base sequence with a high proportion of guanosine residues. Here, the G-quadruplex refers to a unique cubic DNA structure in which four DNA strands rich in guanosine pair with each other to form a quadruplex in the form of hydrogen bonds, and refers to a square structure formed by four guanine bases closely packed through Hoogsteen hydrogen bonds. The G-quadruplex is very stable under various biochemical conditions, and the directionality within the quadruplex varies. In particular, the cation (mainly K+) in the center of the G-quadruplex contributes to further stabilizing the structure of the G-quadruplex. G-quadruplexes are known to have various biological functions in telomere and promoter regions (Henderson E, et al., Telomeric DNA oligonucleotides form novel intramolecular structures containing guanine-guanine base pairs., Cell (December 1987)). In the present invention, 'guanine quadruplex' can be used interchangeably with 'G-quadruplex', 'G-quadruplex', 'guanine quadruplex', 'G-quadruplex', and 'guanine-quadruplex'. It has been reported that guanine quadruplexes can be formed by nucleic acids composed of not only DNA but also RNA.

[0040] Therefore, in the present invention, the guanine quadruplex may be characterized as being DNA or RNA.

[0041] Guanine quadruplexes are formed primarily at guanine-rich sequence sites (Murat P, Balasubramanian S (April 2014)). However, guanine quadruplexes are not formed at all guanine-rich sequence sites. A method for predicting guanine quadruplex formation ability has been reported previously (Todd AK, Johnston M, Neidle S (2005)). For example, Todd AK, Johnston M et al. reported that d(G 3+ N1-7G3+N1-7G 3+ N1-7G 3+ ) (where the subscript represents the number range of each base, N represents any base, and d is an integer greater than or equal to 1, indicating repetition of the base sequence in parentheses) has been reported as a general pattern for forming guanine quadruplexes.

[0042] Therefore, in the present invention, the guanine quadruplex may be characterized by being formed by a sequence in which three or more consecutive guanines and one to seven arbitrary sequences are repeated one or more times, and preferably may be characterized by including a sequence of 5'-GGGN1GGGN2GGGN3GGG-3' (wherein N1, N2, and N3 are one to four arbitrary sequences).

[0043] In the present invention, the guanine quadruplex may be characterized by, but is not limited to, 5'- TTAGGGTTAGGGTTAGGGTTAGGG-3' (SEQ ID NO: 1). In the present invention, a sequence capable of forming a guanine quadruplex may have uracil (U) positioned at the position of thymine (T) when the nucleic acid is RNA.

[0044] In the present invention, the guanine quadruplex may be included at a concentration of 1 to 30 μM, preferably 5 to 15 μM, and more preferably 10 μM, but is not limited thereto.

[0045] In the present invention, the term 'cofactor' refers to a non-protein organic compound or metal ion required for the activity of an enzyme as a catalyst. A cofactor is required for a guanine quadruplex to catalyze the peroxidation reaction, and a cofactor such as hemin has been reported (Owen J. Einarson, et al., Nucleic Acids Research, Volume 45, Issue 17, 29 September 2017, Pages 9813-9822). In the present invention, the hemin may be included at a concentration of 1 to 300 μM, preferably 50 to 150 μM, and more preferably 100 μM, but is not limited thereto, and may be appropriately selected depending on the type and concentration of the guanine quadruplex.

[0046] The above guanine quadruplex can irreversibly dissociate into single strands at a certain temperature and bind to hemin to form a guanine quadruplex-hemin complex.

[0047] In the present invention, the composition may additionally include a stabilizer for the guanine tetramer. Examples of the stabilizer include potassium ions (K+) or sodium ions (Na+).

[0048] In the present invention, the guanine tetramer-hemin complex may be characterized by having peroxidase activity.

[0049] The term 'peroxidase' of the present invention refers to an enzyme that oxidizes a substrate by catalyzing a reaction that dehydrogenates the substrate using hydrogen peroxide, and the reaction in which hydrogen peroxide is reduced and the substrate is oxidized through the catalytic action of the peroxidase is referred to as a 'peroxidation reaction'.

[0050] In the present invention, the guanine quadruplex-hemin complex may be characterized in that the guanine quadruplex is self-biotinylated upon reaction between hydrogen peroxide and biotinyl tyramide.

[0051] In the present invention, the term 'self-biotinylation' refers to the binding of biotin to a guanine quadruplex when a peroxidation reaction is performed using biotinyl tyramide as a substrate through the peroxidase activity of the guanine quadruplex.

[0052] In one embodiment of the present invention, it was confirmed that the guanine quadruplex-hemin complex reduces hydrogen peroxide through peroxidase activity and simultaneously oxidizes the substrate biotinyl tyramide, thereby self-biotinylating the guanine quadruplex.

[0053] “Biotinyl tyramide” is a compound reported as a substrate of peroxidase, and can be represented by the following chemical formula 1. Biotinyl tyramide can be included at a concentration of 1 to 300 μM, preferably 50 to 150 μM, and more preferably 100 μM, but is not limited thereto, and can be appropriately selected depending on the type and concentration of the cofactor and guanine tetramer.

[0054] [Chemical Formula 1]

[0055]

[0056] The composition for detecting hydrogen peroxide according to the present invention is characterized by including a probe capable of binding to the guanine quadruplex and having a labeling substance bound thereto.

[0057] In the present invention, the term "probe" refers to a single-stranded nucleic acid molecule that specifically binds to a target nucleic acid sequence. The probe can bind to the target nucleic acid sequence through hybridization. In particular, the probe of the present invention may be characterized by specifically binding to the guanine quadruplex.

[0058] In the present invention, the probe may be characterized as being a nucleic acid containing a sequence complementary to the guanine quadruplex.

[0059] In the present invention, the term 'hybridization' means that two single-stranded nucleic acids form a duplex structure by pairing complementary base sequences. In this specification, the hybridization may be used interchangeably with the meaning of 'complementarily binding', etc. The pairing of base sequences is preferably A / T(U) and G / C according to the Watson-Crick model, but is not limited thereto. Hybridization can occur when the complementarity between single-stranded nucleic acid sequences is perfect (perfect match), or can occur even when some mismatched bases exist. The degree of complementarity required for hybridization can vary depending on the hybridization reaction conditions, and the conditions used to achieve a stringent specific level vary depending on the properties of the nucleic acids to be hybridized. For example, the length of the nucleic acid portions to be hybridized, the degree of homology, the nucleotide sequence composition (e.g., GC / AT composition), and the nucleic acid type (e.g., RNA, DNA) are considered in selecting hybridization conditions. An additional consideration is whether the nucleic acid is immobilized on a solid support, such as a filter.

[0060] In one embodiment of the present invention, when the guanine quadruplex is a 5'-UUAGGGUUAGGGUUAGGGUUAGGG-3' sequence of SEQ ID NO: 1, the probe may be characterized by including, but is not limited to, a 5'-CCCTAACCCTAACCCTAACCCTAA-3' sequence of SEQ ID NO: 2.

[0061] In the present invention, the probe can bind to a self-biotinylated guanine quadruplex.

[0062] In the present invention, the probe can bind to a guanine quadruplex that is not self-biotinylated.

[0063] In the present invention, the probe may be characterized by having a labeling substance bound thereto.

[0064] The labeling agent may be, for example, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelate, or an enzyme. The labeling agent that binds to the probe as described above is a technique widely known in the art, and the probe can be labeled using a conventional method.

[0065] In the present invention, the labeling substance may be characterized as being bound to the 3' end of the probe. In one embodiment of the present invention, it has been confirmed that detection of hydrogen peroxide is possible using a probe having a labeling substance bound to the 3' end, but the present invention is not limited thereto.

[0066] In the present invention, the labeling material is FAM (6-carboxyfluorescein), DIG (Digoxigenin), FITC (fluorescein isothiocyanate), Texas red, fluorescein, HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethyl rhodamine, Oregon green, alexa fluor, ROX (6-Carboxyl-XRhodamine), TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), cyanine series dyes and It may be characterized by being at least one selected from the group consisting of thiadicarbocyanine.

[0067] Here, the labeling substance can be detected by reacting a binding agent that induces color development of the labeling substance and checking whether color development or fluorescence is expressed.

[0068] As an example, the labeling substance can be detected through the color development of the gold nanoparticles by using gold nanoparticles fixed to the surface of a substance that binds to the labeling substance.

[0069] The binding product of the above self-biotinylated guanine quadruplex and the probe can be detected by utilizing a ligand that specifically binds to biotin.

[0070] In one embodiment of the present invention, it was confirmed that hydrogen peroxide can be indirectly detected by immobilizing a ligand that specifically binds to biotin on a solid support, attaching a binding product of a self-biotinylated guanine quadruplex and a probe using biotin-ligand interaction, and then detecting the binding product of the self-biotinylated guanine quadruplex and the probe through a labeling substance of the probe.

[0071]

[0072] Hydrogen peroxide detection kit

[0073] The present invention relates, from another aspect, to a kit for detecting hydrogen peroxide comprising the above composition.

[0074] The above description of the composition related to the composition for detecting hydrogen peroxide can be equally applied to the kit according to the present invention.

[0075] The optimal amount of reagent, buffer or reactant used for a particular reaction in the above kit can be determined by one skilled in the art and can be manufactured as a separate package or compartment containing the composition of the probe, ligand, etc. mentioned above.

[0076] In the present invention, the kit may be characterized by further comprising a ligand that specifically binds to biotin.

[0077] In the present invention, the ligand that specifically binds to biotin may be characterized by being at least one selected from the group consisting of avidin, streptavidin, and neutravidin, but is not limited thereto, and any ligand that specifically binds to biotin known in the art may be utilized.

[0078] In one embodiment, the ligand that specifically binds to biotin may be characterized as being immobilized on the surface of a solid support.

[0079] In the present invention, the solid support is intended to provide a space in which a ligand that specifically binds to a specific substance is fixed, and may be a plate, a microplate well, a plastic test tube, a glass bead, a plastic bead, a nitrocellulose membrane, a paper strip, etc., but is not limited thereto, and various solid supports used in immunoassays can be applied to the present invention.

[0080] In one embodiment of the present invention, the binding product of the self-biotinylated guanine quadruplex and the probe binds to a ligand immobilized on the surface of a solid support, thereby enabling indirect detection of hydrogen peroxide.

[0081] In the present invention, the kit may be characterized as being a lateral flow assay (LFA) kit.

[0082] In the present invention, the term "lateral flow analysis" generally refers to an analysis method utilizing antibody-antigen reactions, but is not limited thereto and may include methods utilizing specific intermolecular binding. In particular, in one embodiment of the present invention, a lateral flow analysis method was designed utilizing biotin bound to a guanine quadrupole and a ligand that specifically binds to the biotin, and it was confirmed that hydrogen peroxide can be detected with high sensitivity.

[0083] In one embodiment of the present invention, hydrogen peroxide was detected using a solid support comprising:

[0084] A conjugation pad comprising a primary antibody that specifically binds to a labeling substance;

[0085] A test line containing a ligand that specifically binds to biotin; and

[0086] A control line comprising a secondary antibody that specifically binds to the above primary antibody.

[0087] The above primary antibody may be characterized in that it is not fixed to the conjugation pad. The primary antibody binds to a label and can move along the solid support by capillary action.

[0088] In one embodiment of the present invention, the kit may include a solid support arranged in the order of sample pad-conjugation pad-test line-control line-absorbent pad.

[0089] The conjugation pad contains a primary antibody that specifically binds to the label, a ligand that specifically binds to biotin is immobilized on the test line, and a secondary antibody that specifically binds to the primary antibody is immobilized on the control line.

[0090] The sample is introduced from the sample pad and moves sequentially by capillary action, and can exhibit a color change through a specific binding reaction at the test line and control line, respectively.

[0091] In the present invention, the term 'antibody' refers to a wide range of proteins or fragments thereof that specifically bind to an antigen, also known as immunoglobulin, and more specifically, it is a concept that includes not only complete antibodies but also antigen binding fragments of antibodies, including Fab, Fab', F(ab')2, scFv, (scFv)2, scFv-Fc, and Fv, etc.

[0092] The above primary antibody may be characterized by specifically binding to a labeling substance present in the sample to be analyzed, and the type thereof is not particularly limited as long as it is known in the art to specifically bind to the labeling substance.

[0093] The above secondary antibody may be characterized by specifically binding to the above primary antibody, and its type is not particularly limited as long as it is known in the art to specifically bind to the above primary antibody.

[0094] In one embodiment of the present invention, the ligand that specifically binds to biotin may be characterized as being immobilized on the test line. The ligand binds to biotin and can capture the binding product of the self-biotinylated guanine quadruplex and the probe on the test line.

[0095] The probe, the binding product of the probe and the autobiotinylated guanine quadruplex, and the binding product of the probe and the non-autobiotinylated guanine quadruplex all contain the label and can be captured by the control line. Therefore, if the detection kit is not defective, a color change will appear in the control line regardless of the presence or absence of hydrogen peroxide in the sample. The color change in the control line can be used to confirm whether the detection system is functioning properly.

[0096] The above primary antibody may be characterized as a gold conjugated antibody. This induces a color change due to visible light scattering when bound to a label in a sample, thereby generating an indirect detection signal of hydrogen peroxide in the test line and the control line. In addition, the primary antibody may be conjugated to fluorescent nanoparticles, quantum dots, enzymes (e.g., HRP, alkaline phosphatase), magnetic nanoparticles, metal nanoclusters, luminescent nanoparticles, etc., and thus may generate detection signals in various ways, such as fluorescence emission, chemiluminescence, electrochemical signals, and magnetic detection.

[0097] In the present invention, the solid support may further include an absorbent pad that absorbs the remaining sample. The liquid sample can be absorbed through the absorbent pad by capillary action.

[0098]

[0099] Hydrogen peroxide detection method

[0100] From another aspect, the present invention relates to a method for detecting hydrogen peroxide comprising the following steps:

[0101] (a) a step of adding the composition to a sample containing hydrogen peroxide and causing a reaction; and

[0102] (b) a step of detecting a binding product of the self-biotinylated guanine quadruplex and the probe.

[0103] The above description regarding the detection of hydrogen peroxide can be equally applied to the hydrogen peroxide detection method according to the present invention.

[0104] In the present invention, the term "sample" includes various samples, and preferably, a biological sample is analyzed using the method of the present invention. More preferably, it may be a sample mixed with a virus species or a sample of an individual infected with the virus (e.g., a human, a mammal, a fish, etc.), and biological samples of plant, animal, human, fungal, bacterial, and viral origin may be analyzed. When analyzing a sample of mammalian or human origin, the sample may be derived from a specific tissue or organ. Representative examples of tissues include connective, skin, muscle, or nervous tissue. Representative examples of organs include the eye, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, small intestine, testis, ovary, uterus, rectum, nervous system, gland, and internal blood vessels. The biological sample to be analyzed includes any cell, tissue, fluid, or any other medium derived from a biological source that can be well analyzed by the present invention. Additionally, the biological samples being analyzed include body fluid samples, including but not limited to blood, serum, plasma, lymph, breast milk, urine, feces, ocular fluid, saliva, semen, brain extracts (e.g., brain pulverize), spinal fluid, appendix, spleen, and tonsil tissue extracts.

[0105] In the present invention, when hydrogen peroxide is present in the sample, the guanine quadruplex may be characterized in that it is self-biotinylated in step (a).

[0106] In the present invention, the step (a) may be characterized in that the guanine quadruplex is not self-biotinylated when hydrogen peroxide is not present in the sample.

[0107] In the present invention, when hydrogen peroxide is present in the sample, the guanine quadruplex may be characterized in that in step (a), the guanine quadruplex is self-biotinylated, and the self-biotinylated guanine quadruplex binds to the probe.

[0108] In the present invention, when hydrogen peroxide is not present in the sample, it may be characterized in that the guanine quadruplex is not self-biotinylated in the step (a), and the non-self-biotinylated guanine quadruplex binds to the probe.

[0109] In the present invention, the step (a) may be characterized by reacting for 1 minute to 60 minutes, 10 minutes to 50 minutes, preferably 15 minutes to 45 minutes, 20 minutes to 40 minutes, more preferably 25 minutes to 35 minutes, and most preferably 30 minutes, but is not limited thereto.

[0110] In the present invention, the step (b) may be characterized in that it is performed through a lateral flow analysis method using a ligand that specifically binds to biotin.

[0111]

[0112] In one embodiment of the present invention, the method for detecting hydrogen peroxide may include the steps of immobilizing a ligand that specifically binds to biotin on a solid support; adding the composition to a sample to react, and then contacting the same with the solid support.

[0113] In another embodiment, the hydrogen peroxide detection method may be characterized by adding the composition to a sample, causing a reaction, and then applying the composition to a solid support comprising i) a conjugation pad comprising a primary antibody that specifically binds to a label; ii) a test line comprising a ligand that specifically binds to biotin; and iii) a control line comprising a secondary antibody that specifically binds to the primary antibody.

[0114] Here, the binding product of the auto-biotinylated guanine quadruplex and the probe can be captured on the test line, and the binding product of the non-auto-biotinylated guanine quadruplex and the probe and the primary antibody included in the conjugation pad can be captured on the control line.

[0115]

[0116] Detection method of biomolecules

[0117] The present invention relates, from another aspect, to a method for detecting a biomolecule capable of generating hydrogen peroxide by an oxidation reaction comprising the following steps:

[0118] (a) A step of adding an oxidizing enzyme to a sample containing a biomolecule capable of generating hydrogen peroxide through an oxidation reaction and causing a reaction;

[0119] (b) a step of adding a composition according to any one of claims 1 to 7 to the sample and causing a reaction; and

[0120] (c) A step of detecting a biomolecule capable of generating hydrogen peroxide by oxidation reaction by detecting a binding product of a self-biotinylated guanine quadruplex and a probe.

[0121] The above biomolecule may be characterized by being capable of generating hydrogen peroxide through an oxidation reaction.

[0122] The above description regarding the detection of hydrogen peroxide can be equally applied to the method for detecting biomolecules according to the present invention. In addition, steps (a) and (b) of the method for detecting hydrogen peroxide correspond to steps (b) and (c) of the method for detecting biomolecules, respectively, and the description of steps (a) and (b) of the method for detecting hydrogen peroxide can be equally applied to steps (b) and (c) of the method for detecting biomolecules.

[0123] In the present invention, the biomolecule may be characterized by being at least one selected from the group consisting of choline, ethanol, glucose, lactate, xanthine, cholesterol, vitamin C (L-ascorbic acid), amino acid, and monoamine, but is not limited thereto, and a biomolecule capable of generating hydrogen peroxide by an oxidation reaction is included in the scope of the present invention.

[0124] In the present invention, the step (a) may be characterized in that it is performed by adding an oxidizing enzyme that oxidizes the biomolecule.

[0125] Specifically, when the biomolecule is choline, choline oxidase (EC number 1.1.3.17) that oxidizes choline can be utilized. When the biomolecule is ethanol, alcohol oxidase (EC number 1.1.3.13) that oxidizes ethanol can be utilized. When the biomolecule is glucose, glucose oxidase (EC number 1.1.3.4) that oxidizes glucose can be utilized. When the biomolecule is lactate, lactate oxidase (EC number 1.1.3.2) that oxidizes lactate can be utilized. When the biomolecule is xanthine, xanthine oxidase (EC number 1.17.3.2) that oxidizes xanthine can be utilized. When the biomolecule is cholesterol, cholesterol oxidase (EC number 1.1.3.6) that oxidizes cholesterol can be utilized. If the biomolecule is vitamin C, L-ascorbate oxidase (EC number 1.10.3.3) that oxidizes vitamin C can be utilized. If the biomolecule is an amino acid, L-amino acid oxidase (EC number 1.4.3.2) that oxidizes amino acids can be utilized. If the biomolecule is a monoamine, monoamine oxidase (EC number 1.4.3.4) that oxidizes monoamines can be utilized.

[0126]

[0127] Hereinafter, the present invention will be described in detail with examples and the like to aid understanding. However, the examples according to the present invention may be modified in various different forms, and the scope of the present invention should not be construed as being limited to the following examples.

[0128]

[0129] Example

[0130] Example 1. Establishment of SelBio-LFA reaction conditions for H2O2 detection and quantification.

[0131] The preparation process of reaction solution A and reaction solution B for H2O2 detection and quantification is as follows, but is not limited thereto. Reaction solution A contains 3 μL of G-quadruplex (10 μM), 2 μL of hemin (100 μM), 2 μL of biotinyl tyramide (100 μM), 2 μL of HEPES (200 mM, pH 7.4), 1.3 μL of NaCl (3 M), 0.4 μL of KCl (1 M), 2.3 μL of DW, and 25 μL of H2O of various concentrations. Reaction solution B contains 0.6 μL of DP (10 μM), 80 μL of Tris-buffered saline, and 1.4 μL of DW. The prepared reaction solution A was stored at room temperature for 30 minutes to induce self-biotinylation reaction of G-quadruplex. Afterwards, reaction solution A was mixed with reaction solution B, and the mixture was stored at room temperature for 3 minutes, and the red band signal was analyzed using an LFA strip. To analyze the red band signal, a photograph of the LFA strip was taken, and the intensity of the red band signal was measured using the imageJ analysis tool.

[0132] The nucleic acid sequences used in this example are shown in Table 1 below.

[0133]

[0134] Example 2. Verification of the sensitivity and specificity of H2O2 detection using SelBio-LFA technology.

[0135] A sensitivity verification experiment of the present technology was conducted using the reaction conditions mentioned in Example 1. After preparing analytical samples containing various concentrations of H2O2, the SelBio-LFA technology was used to analyze them. As a result, it was confirmed that a red band signal was distinguishable with the naked eye up to 1 μM, and the quantifiable limit of detection (LOD) was 0.388 μM. (Fig. 3) In addition, instead of the target H2O2 contained in the reaction solution A used in Example 1, a reaction solution A containing 400 μM of non-target substances MgCl2, NH4Cl, phenylalanine, cysteine, tryptophan, and glucose was prepared, and the specificity of the present technology was verified through the SelBio-LFA reaction. As a result, it was confirmed that a red band signal was observed on the test line of the LFA strip only in the analytical samples containing H2O2. (Fig. 4)

[0136]

[0137] Example 3. Verification of the feasibility and sensitivity of choline detection using SelBio-LFA technology.

[0138] In order to verify the feasibility of using the SelBio-LFA technology according to the present invention, an experiment was conducted to verify the detectability and sensitivity of choline, a target substance other than H2O2. For this purpose, instead of the reaction solution A used in Example 1, reaction solution A' containing 0.5 μL of choline oxidase (10 U / mL), 3 μL of G-quadruplex (10 μM), 2 μL of hemin (100 μM), 2 μL of biotinyl tyramide (100 μM), 2 μL of HEPES (200 mM, pH 7.4), 1.3 μL of NaCl (3 M), 0.4 μL of KCl (1 M), 6.3 μL of DW, and 0.5 μL of choline at various concentrations was used. As a result of analysis using the above reaction solution A' using the SelBio-LFA technology, it was confirmed that the red band signal was visually distinguishable up to 0.5 μM, and the quantifiable detection limit was 0.226 μM. (Fig. 5)

[0139]

[0140] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0141]

[0142] The detection method using the composition for detecting hydrogen peroxide according to the present invention is simple and easy for anyone to use, while providing high sensitivity and allowing detection of hydrogen peroxide with the naked eye, making it useful in the field of point-of-care testing (POCT). Furthermore, it is capable of detecting various biomolecules that can generate H2O2 products through oxidation reactions, such as choline, ethanol, glucose, lactate, xanthine, cholesterol, and vitamin C, thus demonstrating high applicability.

[0143]

[0144] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0145]

[0146] Electronic file attached.

Claims

1. A composition for detecting hydrogen peroxide comprising: i) G-quadruplex-hemin complex; ii) biotinyl tyramide; and iii) A probe capable of binding to the above guanine quadruplex.

2. A composition for detecting hydrogen peroxide, characterized in that the guanine quadruplex-hemin complex in the first paragraph has peroxidase activity.

3. A composition for detecting hydrogen peroxide, wherein the guanine quadruplex-hemin complex in the first paragraph is characterized in that the guanine quadruplex is self-biotinylated when hydrogen peroxide and biotinyl tyramide are reacted.

4. A composition for detecting hydrogen peroxide, characterized in that the guanine quadruplex in the first paragraph is DNA or RNA.

5. A composition for detecting hydrogen peroxide, characterized in that the probe in the first paragraph is a nucleic acid containing a sequence complementary to the guanine quadruplex.

6. A composition for detecting hydrogen peroxide, characterized in that the probe in the first paragraph has a labeling substance bound thereto.

7. A composition for detecting hydrogen peroxide, characterized in that the labeling substance in the 6th paragraph is bound to the 3' end of the probe.

8. In paragraph 6, the labeling material is FAM (6-carboxyfluorescein), DIG (Digoxigenin), FITC (fluorescein isothiocyanate), Texas red, fluorescein, HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethyl rhodamine, Oregon green, alexa fluor, ROX (6-Carboxyl-XRhodamine), TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), cyanine series dyes and A composition for detecting hydrogen oxide, characterized in that it comprises at least one selected from the group consisting of thiadicarbocyanine.

9. A kit for detecting hydrogen peroxide comprising a composition according to any one of claims 1 to 8.

10. A kit for detecting hydrogen peroxide, characterized in that the kit further comprises a ligand that specifically binds to biotin in the 9th paragraph.

11. A kit for detecting hydrogen peroxide, characterized in that the ligand that specifically binds to biotin in the 10th paragraph is at least one selected from the group consisting of avidin, streptavidin, and neutravidin.

12. A kit for detecting hydrogen peroxide, characterized in that the kit in claim 9 is a lateral flow analysis kit.

13. A method for detecting hydrogen peroxide comprising the following steps: (a) a step of adding a composition according to any one of claims 1 to 8 to a sample containing hydrogen peroxide and causing a reaction; and (b) A step of detecting hydrogen peroxide by detecting the binding product of the auto-biotinylated guanine quadruplex and the probe.

14. A method for detecting hydrogen peroxide, characterized in that in the 13th paragraph, when hydrogen peroxide is present in the sample, the guanine quadruplex is self-biotinylated in step (a), and the self-biotinylated guanine quadruplex binds to the probe.

15. A method for detecting a biomolecule capable of generating hydrogen peroxide by an oxidation reaction comprising the following steps: (a) A step of adding an oxidizing enzyme to a sample containing a biomolecule capable of generating hydrogen peroxide through an oxidation reaction and causing a reaction; (b) a step of adding a composition according to any one of claims 1 to 8 to the sample and causing a reaction; and (c) A step of detecting a biomolecule capable of generating hydrogen peroxide by oxidation reaction by detecting a binding product of a self-biotinylated guanine quadruplex and a probe.

16. A method for detecting a biomolecule, characterized in that in paragraph 15, the biomolecule is at least one selected from the group consisting of choline, ethanol, glucose, lactate, xanthine, cholesterol, vitamin C (L-ascorbic acid), amino acid, and monoamine.

17. A method for detecting a biomolecule, characterized in that in the 15th paragraph, when a biomolecule is present in the sample, the biomolecule is oxidized to generate hydrogen peroxide in step (a), the guanine quadruplex is self-biotinylated in step (b), and the self-biotinylated guanine quadruplex binds to a probe.

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