200934874 九、發明說明： 【發明所屬之技術領域】 本發明係一種目標分子檢測方法，尤指一種利用核 適體與核適體-鉑複合物，以取代習用酵素免疫檢測法 中第一、第二抗體及酵素之使用，進而提供一經濟且穩 定之目標分子檢測方法。 【先前技術】 按，目前在臨床檢測、新藥開發、食物檢測及環境 ❹ 研究等領域中’對於目標分子進行定性定量檢測時，普 遍使用一酵素免疫檢測法（Enzyme Linked immun〇200934874 IX. Description of the invention: [Technical field of the invention] The present invention relates to a target molecule detection method, in particular to a nuclear aptamer and a nuclear aptamer-platinum complex, which replaces the first and the first in the immunoassay of conventional enzymes. The use of diabodies and enzymes provides an economical and stable method of detecting target molecules. [Prior Art] According to the current research, in the fields of clinical testing, new drug development, food testing and environmental ❹ research, when using qualitative and quantitative detection of target molecules, an enzyme immunoassay (Enzyme Linked immun〇) is commonly used.
Sorbent Assay ， ELISA)。 惟’該酵素免疫檢測法具有下列缺點： 一、酵素免疫檢測法中所使用之第一抗體（primary antibody)與第二抗體（sec〇ndary ant丨b〇dy)， 其來源均有其限制且品質純度不一。 一酵素免疫檢測法中所使用之抗體價袼相當昂貴、且 ❹ 無法重覆使用。 三、 酵素免疫檢測法之操作環境多為一生理環境，而抗 體因其適應環境範圍較窄、所以性質較不穩定。 要低皿環境保存、且其活性亦會隨時間慢慢衰退 另外，常因保存條件改變及不同之人員操作，導 四、 酵素免疫檢測法中使用之酵素係為一蛋白質 '其需 另外， 檢測結果之誤差。 ，導致Sorbent Assay, ELISA). However, the enzyme immunoassay has the following disadvantages: 1. The primary antibody and the secondary antibody (sec〇ndary ant丨b〇dy) used in the enzyme immunoassay have their limitations. The quality is of different quality. The price of an antibody used in an enzyme immunoassay is quite expensive and cannot be reused. Third, the operating environment of the enzyme immunoassay is mostly a physiological environment, and the antibody is less stable due to its narrower adaptation environment. It should be stored in a low dish environment, and its activity will gradually decline with time. In addition, due to changes in storage conditions and different personnel operations, the enzyme used in the enzyme immunoassay is a protein. The error of the result. ,resulting in
亟待加以改良。 200934874 【發明内容】 有鑒於上述習用以酵素免疫檢測法對目標分子之 檢測，其有抗體來源之限制且品質純度不一、抗體價格 昂貴且無法重覆使用、抗體適應環境範圍窄、使用之酵 素保存不纽活性麟間衰料缺點；0此發明人依 據多年來從事此課題之相關經驗，乃經過長久努力研究 與實驗，並配合相關學理，終於開發設計出本發明之一 種「目標分子檢測方法」。 Ο ❹ 本發明之一目的，在於提供一種目標分子檢測方 法，該方法係利用—核適體與目標分子之間的專一性鍵 結特，來檢測，該檢測之目標分子可為絲酸、蛋白 質藥物’有機分子及無機分子，亦可廣泛制於臨床 檢測、新藥開發、食物檢測、基因檢測、及環境研究等 i目關之分子生物領域，藉以取代制酵素免疫檢測法中 抗體使用，進而解決該抗體之來源限制、品質純度不 —且價格昂貴之缺點。 古本發明之另-目的’在於提供—種目標分子檢測 ’，該方法係利用一核適體一鉬複合物，以取代習用 培疫檢麻巾之酵素制，藉贿決其於使用環 竟中性質極不穩定且不易保存之缺點。 【實施方式】 浦手段及運 w 、瞭解，缝舉一實施例配合圖 式’坪細說明如下。 本發明係-種「目標分子檢測方法」，尤指一種利 200934874 用一核適體與一核適體-鉑複合物，以取代習用酵素免 疫檢測法中抗體及酵素之使用，藉以提供一種經濟且穩 定之目標分子檢測方法；請參閱第一圖所示，該方法係 包括以下步驟： 步驛一.製備一分析物(analyte)溶液100"1 ; 步驟二：將該分析物溶液置於一載體（如：微孔 盤）中，且該微孔盤中固定有一核適體（在本發明中該 核適體係為一捕捉核適體（Capture apfamer)如一 O biotinylated核適體），以形成一第一混合溶液並於室 溫下反應1小時； 步驟三：以一洗滌液（在本發明中該洗滌液為一 Tris-HCl溶液）300y 1洗滌該第一混合溶液3次； 步驟四：加入一核適體-鉑複合物（在本發明中該 核適體-麵複合物係為經一銘修飾過之一標定核適體 (probe aptamer)如一鉑（pt)-去氧核醣核酸（dna) 複合物（complex)) 1〇〇μι於該第一混合溶液中，並 ^ 於室溫下反應1小時； 步驟五：再以該洗滌液3〇〇//1洗滌該第一混合溶 液3次； 步驟六：加入一基質（在本發明中該基質為一四 甲基 聯笨胺 (3, 3，5, 5 -tetramethylbenzidine，ΤΜΒ))與一氧化 劑（在本發明中該氧化劑為一過氧化氫（hydr〇gen peroxide))所形成之一第二混合溶液丨00(該第二 混合谷液顏色為澄清透明，若遇·一目標分子，其顏色 200934874 會轉變為淡黃色）於該第一混合溶液中，以形成一第 三混合溶液，並在避光及室溫之環境條件下反應30分 A»A. · 鐘， 步驟七：再加入一終止液1〇〇//1 (在本發明中該 終止液為一磷酸（H3P〇4)溶液）於該第三混合溶液中； 步驟八：判斷（在本發明中該判斷方式係選自肉眼 及一酵素免疫分析儀（Enzyme Linked Immuno Sorbent Assay reader, ELISA-reader)其中之一）該第三混合 © 溶液之顏色是否由澄清透明變為淡黃色？若是，即進入 步驟九’若無，即進入步驟十； 步驟九：表示該分析物溶液中有一目標分子存在。 步驟十：表示該分析物溶液中沒有該目標分子存 在。 在本發明中，該目標分子係選自一胺基酸、一胜 肽、一蛋白質、一藥物、一無機小分子及一細胞其中之 —〇 〇 在本發明中，固定該核適體於該載體上之作用力 為一共價鍵結作用力。 在本發明中，該鉑係包含任何形式之一鉑鹽複合 物。 在本發明中，該第二混合液係包含所有可被一辣 根過氧化物酶（Horseradish peroxidase)及該核適體一 銘複合物所催化呈色之溶液。 人在本發明中，該終止液為一可改變該核適體—鉑複 Q物與該基質及該氧化劑之反應環境條件（如酸驗值） 200934874 之溶液（如—磷酸（Η3Ρ〇4)溶液）。 於：由上述可知’本發明之方法有別習用技術關鍵在 一、 方f具有新穎性及進步性，係利用核適體 :所：r抗趙及!素，進而解決其== ❹ Ο ，丨@,1®=錢範圍*、不胃保存及其活性會隨時間 _又&城之缺點，故具有卿性及進步性。 二、 法具有實用性，係利用合成核適體與 鉑複合物，以解決習用酵素免疫檢測法中 Ά體與酵素價格昂貴之缺點，故有其實用性。 —按，上述詳細說明為針對本發明之一種較佳之可 例說明而已’惟該實施例並_赚定本發明之 请專利麵’舉凡其他未麟本㈣賴示之技藝精 下所完成之均等變化與修飾變更，均應包含於本發明 所涵蓋之專利範圍中。 【圖式簡單說明】 第一圖係為本發明之流程圖。 【主要元件符號說明】Urgent to be improved. 200934874 [Summary of the Invention] In view of the above-mentioned enzyme immunoassay for the detection of target molecules, there are restrictions on the source of antibodies and the quality is different, the antibody is expensive and cannot be reused, the antibody is adapted to a narrow environment, and the enzyme is used. The shortcomings of the non-active lining failure material; 0. Based on years of experience in this subject, the inventor has developed and designed a "target molecular detection method" of the present invention through long-term research and experimentation, and with relevant academic principles. "." Ο ❹ An object of the present invention is to provide a method for detecting a target molecule, which is detected by utilizing a specific bond between a nucleus aptamer and a target molecule, and the target molecule of the detection may be silk acid or protein. The drug 'organic molecules and inorganic molecules can also be widely used in the field of molecular biology such as clinical testing, new drug development, food testing, genetic testing, and environmental research, in order to replace the use of antibodies in the enzyme immunoassay. The source of the antibody is limited, the quality of the purity is not - and the price is expensive. Another object of the invention is to provide a target molecule detection method, which utilizes a nucleus aptamer-molybdenum complex to replace the enzyme system of the diagnosing anaesthesia towel, and uses the bribe to determine the use of the ring. The nature is extremely unstable and difficult to save. [Embodiment] The method and operation of the pump and the understanding of the sewing method are described in the following paragraphs. The invention relates to a "target molecule detection method", in particular to a benefit of 200934874 using a nucleus aptamer and a nucleus aptamer-platinum complex to replace the use of antibodies and enzymes in the conventional enzyme immunoassay, thereby providing an economy. And a stable target molecule detection method; see the first figure, the method includes the following steps: Step 1. Prepare an analyte solution 100 "1; Step 2: Place the analyte solution in a a carrier (eg, a microplate) in which a nuclear aptamer is immobilized (in the present invention, the nuclear system is a capture apfamer such as an O biotinylated nuclear aptamer) to form a first mixed solution and reacted at room temperature for 1 hour; Step 3: washing the first mixed solution 3 times with a washing liquid (in the present invention, the washing liquid is a Tris-HCl solution); Step 4: Adding a nuclear aptamer-platinum complex (in the present invention, the nuclear aptamer-face complex is one of the modified aptamers such as a platinum (pt)-deoxyribonucleic acid (A Platinum (pt)-deoxyribonucleic acid) Dna) complex (complex) 1〇〇 Ii in the first mixed solution, and reacted at room temperature for 1 hour; Step 5: further wash the first mixed solution 3 times with the washing liquid 3 〇〇 / / 1; Step 6: add a substrate (in In the present invention, the substrate is a tetramethylbenzidine (3,3,5,5-tetramethylbenzidine, oxime) and an oxidizing agent (in the present invention, the oxidizing agent is hydroxide) Forming a second mixed solution 丨00 (the color of the second mixed gluten solution is clear and transparent, if a target molecule, its color 200934874 will change to pale yellow) in the first mixed solution to form a third Mix the solution and react in the dark and room temperature conditions for 30 minutes A»A. · Clock, Step 7: Add another stop solution 1〇〇//1 (In the present invention, the stop solution is monophosphate ( H3P〇4) solution) in the third mixed solution; Step 8: Judging (in the present invention, the judgment method is selected from an Enzyme Linked Immuno Sorbent Assay reader (ELISA-reader)) a) Whether the color of the third mixed solution is clear Ming becomes pale yellow? If yes, proceed to step IX. If not, proceed to step 10; Step IX: indicates that a target molecule is present in the analyte solution. Step 10: indicates that the target molecule is absent in the analyte solution. In the present invention, the target molecule is selected from the group consisting of an amino acid, a peptide, a protein, a drug, an inorganic small molecule, and a cell thereof. In the present invention, the nuclear aptamer is immobilized thereon. The force on the carrier is a covalent bonding force. In the present invention, the platinum group comprises a platinum salt complex in any form. In the present invention, the second mixed solution contains all solutions which can be colored by a horseradish peroxidase and a complex of the nuclear aptamer. In the present invention, the terminating solution is a solution (for example, -phosphoric acid (Η3Ρ〇4)) which changes the reaction environment condition (such as acid value) of the nuclear aptamer-platinum complex Q with the substrate and the oxidizing agent. Solution). It can be seen from the above that the method of the present invention has the key to the use of the technology, and the use of the nuclear aptamer: r: anti-Zhao and !, and then solves its == ❹ Ο,丨@,1®=Money range*, non-stomach preservation and its activity will be clear and progressive over time _ and & Second, the method is practical, and it uses the synthetic aptamer and platinum complex to solve the shortcomings of the expensive enzymes and enzymes in the immunoassay of the conventional enzyme, so it has its practicability. - The above detailed description is directed to a preferred embodiment of the present invention and has been described as an example of the present invention and the invention of the invention is not limited to the scope of the invention. Modifications and modifications are intended to be included in the scope of the patents covered by the invention. BRIEF DESCRIPTION OF THE DRAWINGS The first figure is a flow chart of the present invention. 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