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Electrochemiluminescence detection method for nucleic acid using intercalatorRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidElectrochemiluminescence detection method for nucleic acid using intercalator description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060292595, Electrochemiluminescence detection method for nucleic acid using intercalator. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This is a Divisional Application of U.S. application Ser. No. 10/282,251 filed Oct. 29, 2002, the entirety of which is incorporated herein by reference. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates to a method for detecting nucleic acid hybridization, in which electrochemiluminescence is promoted by using intercalator which is peculiarly binding only to double-stranded nucleic acid. [0004] 2. Description of the Background Art [0005] Methods for detecting a result of nucleic acid hybridization generally include nucleic acid hybridization detection methods by a radioautography, a laser-induced fluorescence (LIF) and an electrochemical method. [0006] The radioautography method which detects nucleic acid hybridization by labeling target nucleic acid as radioactive isotope is the most widely used in molecular biology. .sup.32P is used as the radioactive isotope, and photographic films are used to detect a binding state of target oligonucleotide and probe oligonucleotide which are labeled. The radioautography method does not require much basic knowledge, thereby easily being applied. However, the radioautography method has several disadvantages that long analysis time such as several hours and a day makes it impossible to know a result fast, a resolution ability is low with orders of 0.1.about.10 .mu.m, and radioactive isotope used in excitation is not stable. [0007] Recently, a laser-induced fluorescence (LIF) method is much used in DNA hybridization detection because several kinds of fluorescence material is used, a resolution ability is excellent, and a result is immediately known. Nowadays, if a charge coupled device (CCD) camera to which fluorescence analysis and image technique are combined is introduced, molecules labeled as fluorescence materials can be imaged real time. However, said method also has several disadvantages that DNA of a sample has to be labeled as fluorescence material before measuring the DNA of a sample, a process to separate and refine the DNA is complicated, a stability for experiments is required, and expensive equipments such as a laser and an attached device for optical detection and an expensive image scanner for scanning a two-dimensional substrate are required. [0008] The method for detecting DNA hybridization by an electrochemical method is one to detect DNA hybridization by using a binding of metallic complex having activity electrochemically and double-stranded DNA. Even if the method is enough simple to provide a cheap detection apparatus, sensitivity is not good. [0009] Since the aforementioned conventional methods for detecting nucleic acid hybridization have several disadvantages, a new detecting method having a high sensitivity is necessary. Especially, a development of a small and cheap system for detecting nucleic acid fast without a process that a sample is bound to label material is required as a portable diagnosis device. SUMMARY OF THE INVENTION [0010] Therefore, an object of the present invention is to provide a method for detecting nucleic acid hybridization having simplification and excellent detection sensitivity without a separate labeling process, in which electrochemiluminescence is caused by inducing an oxidation-reduction reaction between intercalator and transition metallic complex. [0011] An object of the present invention is to provide a method for detecting nucleic acid hybridization, comprising steps of applying proper voltage and using intercalator of nucleic acid to induce an electrochemiluminescence reaction of transition metallic complex; and detecting the quantity of light generated thereby, wherein the intercalator peculiarly binds to double-stranded nucleic acid which is formed by hybridization between probe nucleic acid fixed on a metal surface and target nucleic acid in solution and causes an electrochemiluminescence reaction by inducing an oxidation-reduction reaction of transition metallic complex. The above intercalator can be selected is from the group consisting of doxorubicin, daunorubicin and DAPI(4',6-Diamidino-2-phenylindole). [0012] Another object of the present invention is to provide a novel use of DAPI as an effective intercalator, The foregoing and other objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of the present invention when taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS [0013] The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention. [0014] In the drawings: [0015] FIG. 1 shows a principle of electrochemiluminescence by intercalator, in which I indicates a working electrode; [0016] FIGS. 2 shows a hybridization process between probe nucleic acid fixed on a gold plate and target nucleic acid, and a principle of electrochemiluminescence by intercalator, in which 1 indicates a working electrode, 2 indicates a gold plate, and 3 indicates double-stranded nucleic acid; [0017] FIG. 3 shows chemical structures of Tris(2,2'-bipyridyl)ruthenium (II) [Ru(bpy).sub.3.sup.2+] and Tris(1,10-phenanthroline)ruthenium(II) [Ru(phen).sub.3.sup.2+], corresponding to transition metallic complex; [0018] FIG. 4 shows chemical formulas of doxorubicin and daunorubicin, corresponding to intercalator; [0019] FIG. 5 shows a hybridization detection result of nucleic acid using doxorubicin and Ru(bpy).sub.3.sup.2+ solution, in which a indicates quantity of light detected at a bare gold plate state, b indicates quantity of light detected when probe nucleic acid is fixed on the gold plate, c indicates quantity of light detected when probe nucleic acid is hybridized with target nucleic acid, and d indicates quantity of light detected when doxorubicin is added to the hybridized nucleic acid; [0020] FIG. 6 shows a hybridization detection result of nucleic acid using doxorubicin and Ru(bpy).sub.3.sup.2+ solution, in which e indicates quantity of light detected after reacting doxorubicin with probe nucleic acid and then washing, f indicates quantity of light detected after processing doxorubicin to the hybridized nucleic acid and then washing; and [0021] FIG. 7 shows a hybridization detection result of nucleic acid using daunorubicin and Ru(bpy).sub.3.sup.2+ solution, in which g indicates quantity of light detected after reacting daunorubicin with probe nucleic acid and then washing, h indicates quantity of light detected after processing daunorubicin to the hybridized nucleic acid and then washing. 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