TW201043701A - A gold nanoparticles' hybrid chromatography for DNA molecules detection - Google Patents

Abstract

The invention provides a hybrid chromatography with gold nanoparticles as signal for DNA detection. In this assay, two single-stranded DNA probes were complementary to target DNA molecules, wherein one probe was combined to gold nanoparticles and another probe was combined to biotin. After the probes complementarily annealed to the single strand target DNA, the avidin was added to bind to the biotin probe. The complex containing DNA hybrid, gold nanoparticle, and avidin migrated to the test zone where an anti-avidin antibody was immobilized on the cellulose ester membrane and catched the complex. Consequently, a red line formed because of gold nanoparticles. This DNA molecule detection method which detects whether the target DNA molecules present in the sample is simple and fast.

Description

201043701 、發明說明： 【發明所屬之技術領域】 本發明係關於一種偵測DNA分子之方法，更具體而言 係為一種用於偵測DNA分子之金奈米粒子雜交層析法，係 利用與目標DNA分子互補之兩探針，且該等探針係分別與 金奈米粒子及生物素結合，當兩探針與目標DNA互補黏合 後，利用抗生物素蛋白及抗-抗生物素蛋白抗體，進行相關 之雜交反應，可於硝化纖維膜上藉由該金奈米粒子之呈色 來檢測出目標DNA分子，係為一種簡單、迅速偵測DNA 分子之方法。 【先前技術】 現今臨床醫學、環境衛生、生醫組織工程、農漁畜牧 以及食品微生物安全檢驗等等的領域中，如何迅速、正確 的檢驗分辨出樣本中之DNA分子，是非常重要的，因為檢 驗測試若是有任何差錯，或是檢驗報告作業時間過於冗 長，係會影響後續程序動作的結果，例如防治措施、食品 管制或是醫學治療等等，將會大大的影響到人類的生命安 全、身體健康、食品安全及生活品質，所以發展出一種可 迅速及準確檢驗出細菌種類之方法是有需要的。 除了快速、正確之外，通常還會要求該方法執行和製 作的成本低廉，以及符合簡單、安全等等的性質，才能被 廣範地使用，而且其操作最好是不須經過特別的專業特訓 以及有諸多安全上的顧慮。 201043701 本發明之一種用於偵測dna分子之金奈米粒子雜交 層析法’常見的檢驗測試DNA分子的方法為：聚合酶鏈式 反應（PCR)檢測、DNA-RNA hybrid-capture test 及 DNA chip 等’其中以DNA chip可有效檢測出臨床醫學上各種疾病之 型別’諸如可檢驗出人類乳突瘤病毒(Human papillomavirus， HPV)之型別’亦可藉由該dna chip之檢測而判定該病患 是否為高危險型’亦可藉由不同時期之檢測而判斷病患是 否為不同型別HPV之短暫感染’或是同一型別HPV之永 ❹ 續感染，然而，該DNA chip之檢驗卻是最為昂貴。 而以PCR方式針對DNA分子進行檢測是非常普遍 的，該方法係針對所欲分辨目標DNA分子而設計引子 (primer)，其後經過一些操作程序’而檢測出目標dna分 子，此一方法相較於一些傳統檢測方法具專一性及高靈敏 度，但是，從事PCR之操作人員亦需要經過一定的訓練， 即便s亥PCR操作簡便’但於操作pCR時另一重問題就是污 Ο 染，因為pcr·係藉由引子dna片段與特定目標基因DNA 片段間：變性(denaturation)、引子附著（annealing)、聚合化 (polymeration)反覆藉由聚合酶完成3〇次左右之DNA放 大，其間於原始模板(temple)若有所污染，將導致整個檢結 果之正確性，所以操作人員須相#的知，否則就會整個 檢驗將失去其正確性。再者，PCR檢測成本並不低，它所 使用的儀器設備及所需要引子等等花費都不為少數，從而 降低其使用的普及率：此外，PCR檢測所需要耗費的時間 亦是大問題’即使它比-些傳統檢測方法所需時間還要 201043701 短’但僅就該聚合酶鏈合反應之時間就需一個早上或一個 下午’更不用說其他的結果分析及樣本準備時間。 而 DNA_RNA hybrid-capture test 係為 DNA-RNA 雜交 捕獲系統，其是利用DNA-RNA之雜交後，再以專一性高 的單株抗體捕捉(antibody capture)該DNA-RNA雜交分子， 再藉由化學發光訊號來進行檢測，然而，此方法須利用特 殊的RNA探針來產生出DNA-RNA hybrid，而就習知而 言’ RNA穩定度較差，故此法較不為大眾所採用。 而奈米金粒子在過去幾年來一直被利用於免疫分析實 驗技術上’在 Thanh，Ν· T. K.，Rosenweig，Z.等人於 2002 發 表之 Development of an aggregation-based immunoassay for anti-protein A using gold nanoparticles. Anal. Chem. 74, 1624.文獻中，指出奈米金粒子由於受到表面電漿共振的影 響因此在波長520 nm下會呈現一紅色的色澤而非金黃 色’而此種方法應用於免疫分析上是相當靈敏的。本發明 之發明者利用奈米金粒子探針去黏合目標DNA分子，再藉 由另一探針黏合目標DNA分子並固定於硝化纖維膜上，可 有效债測目標DNA分子，且其靈敏度可到達10 attomoles ’ 係為一新穎、有效、快速的DNA分子檢測法。 繁於上述習知檢測DNA分子之缺點及使用金奈米粒 子檢測法之優點，是以發展出本發明之用於偵測DNA分子 之金奈米粒子雜交層析法：係利用與目標DNA分子互補之 兩探針，且該等探針係分別與金奈米粒子及生物素結合， 虽兩探針與目標DNA互補黏合後，利用抗生物素蛋白及抗 201043701 -抗生物素蛋白抗體，進行相關之雜交反應，可於硝化纖維 膜上藉由該金奈米粒子之呈色來檢測出目標DNA分子，係 為一種簡單、迅速偵測DNA分子之方法。此一方法之優點 在於不但可以在較短的時間内知道測試結果，而且成本低 廉，可以一次處理較多量的樣本；另外其專一性的問題， 由於本發明是利用探針與目標DNA分子之互補黏合原 理，具較高之專一性。 【發明内容】 有鑑於上述習知技術於偵測DNA分子之缺憾，發明人 研發出一種用於偵測DNA分子之金奈米粒子雜交層析 法，其係可有效偵測DNA分子，可節省成本及時間，並具 有高靈敏度’係為一有效之DNA分子檢測方法。 本發明之目的係提供一種用於偵測目標DNA之奈米 金粒子雜交層析法’其係包含下列步驟： a. 將抗-抗生物素蛋白抗體（anti_avidin antib〇dy)固定 於硝化纖維膜上； b. 將待測DNA、金奈米粒子探針及生物素DNA探針 混合，並升高溫度（用於打開待測DNA之雙股，約 啊，2分鐘）後控制溫度（价，不同的探針組; 調整此溫度’以達最佳效果）2Q分鐘，使該等 與單股DNA進行互補黏合（其中該金奈米粒子^針 及該生物素DNA探針係分別與目標dNa同一仰 股互補）； 早 201043701 C.將步驟b之反應液加入抗生物素蛋白（avidin)，使其 與生物素DNA探針結合； d·將步驟c反應液流經步驟a之硝化纖維膜以進行抗_ 抗生物素蛋白抗體針對抗生物素蛋白之捕捉，若有 呈色（紅色）反應’則待測DNA中含有目標DNA。 如上所述之奈米金粒子雜交層析法’其中該目標DNA 為人類乳突瘤病毒DNA之片段。 如上所述奈米金粒子雜交層析法，其中該待測DNA係 為經純化之DNA分子。 如上所述奈米金粒子雜交層析法，其中該目標DNA分 子之單股片段序列為（5’至 3，）：201043701, invention: [Technical Field] The present invention relates to a method for detecting a DNA molecule, and more particularly to a hybridization chromatography method for detecting a DNA molecule, which is utilized by Two probes complementary to the target DNA molecule, and the probes are respectively combined with the gold nanoparticles and biotin, and when the two probes are complementary to the target DNA, the avidin and the anti-avidin antibody are utilized. The related hybridization reaction can detect the target DNA molecule by the coloration of the gold nanoparticle on the nitrocellulose membrane, which is a simple and rapid method for detecting the DNA molecule. [Prior Art] In the fields of clinical medicine, environmental sanitation, biomedical tissue engineering, agricultural and fish farming, and food microbiological safety testing, how to quickly and correctly test the DNA molecules in the sample is very important because If there are any errors in the inspection test, or if the inspection report is too long, it will affect the results of subsequent procedures, such as prevention measures, food control or medical treatment, etc., which will greatly affect the safety of human life and the body. Health, food safety and quality of life are therefore needed to develop a method for quickly and accurately detecting bacterial species. In addition to being fast and correct, it is usually required to be inexpensive to implement and produce, and to be simple, safe, etc., to be widely used, and it is best to operate without special special training. And there are many security concerns. 201043701 A gold nanoparticle hybridization chromatography method for detecting DNA molecules of the present invention is a common method for testing DNA molecules: polymerase chain reaction (PCR) detection, DNA-RNA hybrid-capture test and DNA. Chip, etc., in which DNA chip can effectively detect the type of various clinical diseases, such as the type of human papillomavirus (HPV) can be determined by the detection of the DNA chip Whether the patient is of high risk type can also be judged by different periods of time to determine whether the patient is a transient infection of different types of HPV or a persistent infection of the same type of HPV. However, the test of the DNA chip It is the most expensive. It is very common to detect DNA molecules by PCR. This method is designed to design a primer for the target DNA molecule to be resolved, and then to detect the target DNA molecule through some operating procedures. Some traditional detection methods are specific and sensitive. However, the operators who work in PCR also need some training, even if the operation of sHPCR is simple, but another problem in the operation of pCR is contamination, because pcr Between the DNA fragment of the primer and the DNA fragment of the specific target gene: denaturation, annealing, and polymerization, the DNA amplification is performed by the polymerase 3 times or so, in the original template (temple). If there is pollution, it will lead to the correctness of the whole inspection result, so the operator must know the truth, otherwise the whole inspection will lose its correctness. Furthermore, the cost of PCR detection is not low, and the equipment used and the required primers are not limited, which reduces the popularity of the use: In addition, the time required for PCR detection is also a big problem. Even if it is shorter than the time required for some traditional detection methods, 201043701 is 'but only one morning or one afternoon for the time of the polymerase chain reaction', not to mention other results analysis and sample preparation time. The DNA_RNA hybrid-capture test is a DNA-RNA hybrid capture system that utilizes DNA-RNA hybridization and then captures the DNA-RNA hybrid molecule with a highly specific monoclonal antibody, and then chemically Luminescence signals are used for detection. However, this method requires the use of special RNA probes to generate DNA-RNA hybrids, which are conventionally less robust, so this method is less popular. Nanogold particles have been used in immunoassay experimental techniques for the past few years. 'Development of an aggregation-based immunoassay for anti-protein A using gold in Thanh, T. TK, Rosenweig, Z. et al. Anal. Chem. 74, 1624. In the literature, it is pointed out that nanogold particles exhibit a red color at the wavelength of 520 nm instead of golden yellow due to the influence of surface plasma resonance. The analysis is quite sensitive. The inventors of the present invention use the nano gold particle probe to bind the target DNA molecule, and then bind the target DNA molecule by another probe and fix it on the nitrocellulose membrane, thereby effectively measuring the target DNA molecule, and the sensitivity thereof can be reached. 10 attomoles ' is a novel, efficient and rapid DNA molecular assay. The advantages of the above-mentioned conventional detection of DNA molecules and the use of the gold nanoparticle detection method are to develop the hybridization chromatography method of the invention for detecting DNA molecules: the utilization and target DNA molecules Two complementary probes, which are combined with the gold nanoparticles and the biotin, respectively. After the two probes are complementary to the target DNA, the avidin and the anti-201043701-avidin antibody are used. The related hybridization reaction can detect the target DNA molecule by the coloration of the gold nanoparticle on the nitrocellulose membrane, and is a simple and rapid method for detecting the DNA molecule. The advantage of this method is that not only can the test result be known in a short time, but also the cost is low, and a large amount of sample can be processed at one time; in addition, the problem of specificity, since the present invention utilizes the complement of the probe and the target DNA molecule The principle of bonding has a high degree of specificity. SUMMARY OF THE INVENTION In view of the above-mentioned conventional techniques for detecting defects in DNA molecules, the inventors have developed a hybridization chromatography method for detecting DNA molecules, which can effectively detect DNA molecules and saves Cost and time, and high sensitivity 'is an effective method for DNA molecular detection. The object of the present invention is to provide a nanogold hybridization chromatography method for detecting target DNA, which comprises the following steps: a. immobilizing an anti-avidin antibody (anti_avidin antib〇dy) on a nitrocellulose membrane b. Mix the DNA to be tested, the gold nanoparticle probe and the biotin DNA probe, and raise the temperature (used to open the double strands of the DNA to be tested, about 2 minutes) to control the temperature (price, Different probe sets; adjust this temperature to achieve the best effect) 2Q minutes to make these and the single strand of DNA complementary binding (where the gold nanoparticle needle and the biotin DNA probe system respectively and the target dNa The same as the opposite strand); early 201043701 C. The reaction solution of step b is added to avidin (avidin) to bind to the biotin DNA probe; d· the reaction liquid of step c is passed through the nitrocellulose membrane of step a For the capture of avidin antibody against avidin, if there is a coloration (red) reaction, the DNA to be tested contains the target DNA. The nanogold particle hybridization chromatography as described above wherein the target DNA is a fragment of human papillomavirus DNA. The nanogold particle hybridization chromatography as described above, wherein the DNA to be tested is a purified DNA molecule. The nanogold particle hybridization chromatography as described above, wherein the single-stranded fragment sequence of the target DNA molecule is (5' to 3,):

TTCCTCCCCATGTCGTAGGTACTCCTTAAAGTTAGTATT TTTATATGTAGTTTCTGAAGT。 如上所述奈米金粒子雜交層析法，其中該待測DNA之 偵測靈敏度可達amol(lCT18 mol)。 如上所述之奈米金粒子雜交層析法，其中該抗-抗生物 素蛋白抗體(anti-avidin antibody)之濃度為15 pmol/μΐ;該金 奈米粒子探針濃度為0.4 fmol/μΐ ;該生物素DNA探針濃度 為100 pmol/μΐ ;該抗生物素蛋白（avidin)之濃度為15 pmol/μΐ ° 【實施方式】 為使充分暸解本發明之目的、特徵及功效，茲籍由下 述具體之實施例，並配合所附之圖式，對本發明做—詳細 201043701 說明’說明如後： 首先’本發明一種用於偵測DNA分子之金奈米粒子雜 父層析法，其設計原理如第一圖所示，其係於一長條型硝 化纖維臈6上’固定抗-抗生物素蛋白抗體9(anti-avidin antibody)作為測試條帶7，再者，將待測dna分子丨、金 Ο Ο 奈米粒子探針2及生物素DNA探針3之反應液升溫(至約 95 C ’ 2分鐘以使其内之待測dna分子工變性(d咖㈣ 而打開雙股DNA分子成為單股DNA分子4，之後控制溫 度（=5C，不同的探針組可調整此溫度，以達最佳效果） 20分鐘，使該金奈米粒子探針2及該生物素DNA探針3 與該目標單股舰分子4進行互獅合（其巾該金奈米粒 子探針及該生物素DNA探針係分別與目標DNA分子之同 側單股互補），接著將反應後之反舰加人抗生物素蛋白 (avidin)5,使該抗生物素蛋白5與生物素dna探針3 έ士人， =終反應加人已㈣抗·抗生物素蛋白抗體9之確化纖°維 ㈣吸收墊8使該反應液利用毛細現象流經該 j條帶7，此時若待測DNA分子具有目標Dna分子， 、[、係黏合有金奈米粒子探針2及生物素dna探針3 ，物素DNA探針3與抗生物素蛋白5及抗_抗生物素蛋^ 、^ 9連結’使該目標DNA分子被固^肖化纖維膜 =1二再藉由結合該目標DNA分子上之金奈米粒 之測心雉7奈練子之呈色，而於_化纖維暝6上 、# ^、▼產生紅色條帶，得以偵測出該目標八 施例一：以人類乳突病毒檢測對象 9 201043701 i. 偵測HVP之DNA探針 金奈米粒子探針 引子長度：35 bpsTTCCTCCCCATGTCGTAGGTACTCCTTAAAGTTAGTATT TTTATATGTAGTTTCTGAAGT. The nanogold particle hybridization chromatography as described above, wherein the detection sensitivity of the DNA to be tested is up to amol (lCT18 mol). The nano gold particle hybridization chromatography as described above, wherein the concentration of the anti-avidin antibody is 15 pmol/μΐ; the concentration of the gold nanoparticle probe is 0.4 fmol/μΐ; The concentration of the biotin DNA probe is 100 pmol/μΐ; the concentration of the avidin is 15 pmol/μΐ [Embodiment] In order to fully understand the purpose, characteristics and efficacy of the present invention, DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT AND EMBODIMENT OF THE INVENTION The detailed description of the present invention is described in detail as follows: First, the first embodiment of the present invention is a heterogeneous chromatography method for detecting gold molecules of DNA molecules. The principle is as shown in the first figure, which is attached to a long strip of nitrocellulose 臈6 to immobilize anti-avidin antibody as test strip 7, and further, the dna molecule to be tested丨, 金Ο Ο Nanoparticle probe 2 and biotin DNA probe 3 reaction solution is heated (to about 95 C ' 2 minutes to make the DNA molecule to be tested (d coffee (4) and open double-stranded DNA The molecule becomes a single strand of DNA molecule 4, after which the temperature is controlled (=5C, different probe sets can be Adjusting the temperature to achieve the best effect) for 20 minutes, the Jinnai particle probe 2 and the biotin DNA probe 3 are lion-grayed with the target single-stranded molecule 4 (the towel is the gold nanoparticle) The probe and the biotin DNA probe system are complementary to the same side of the target DNA molecule, respectively, and then the anti-ship of the reaction is added with avidin 5 to make the avidin 5 and the organism Prime dna probe 3 gentleman, = final reaction plus human (four) anti-avidin antibody 9 is determined by the chemical fiber ° (4) absorption pad 8 so that the reaction solution through the capillary phenomenon through the j strip 7, at this time If the DNA molecule to be tested has the target DNA molecule, [, the system is bonded with the gold nanoparticle probe 2 and the biotin dna probe 3, the analyte DNA probe 3 and the avidin 5 and the anti-antibiotic egg ^, ^9 linkage 'make the target DNA molecule to be fixed to the fiber membrane = 1 and then bind to the color of the heart of the gold nanoparticle on the target DNA molecule, and then On the fiber 暝6, #^, ▼ produces a red band, which can detect the target. Example 1: Detection of human papillomavirus 9 201043701 i. Detection DNA probe of HVP Jinnai particle probe Lead length: 35 bps

引子序列（5,至 3，）： AAAAAAAAAAACTTCAGAAACPrimer sequence (5, to 3,): AAAAAAAAAAACTTCAGAAAC

TACATATAAAAATA 生物素DNA探針 引子長度：35 bpsTACATATAAAAATA Biotin DNA Probe Primer Length: 35 bps

引子序歹|J(5,至 3，）： biotin- AAAAAAAAAAGGAGTAPrimer 歹|J(5, to 3,): biotin- AAAAAAAAAAGGAGTA

CCTACGACATGGGGAGGAA 目標ssDNA分子（HPV ssDNA片段） 合成序列長度：60 bpsCCTACGACATGGGGAGGAA Target ssDNA molecule (HPV ssDNA fragment) Synthetic sequence length: 60 bps

序列（5’至 3，）： TTCCTCCCCATGTCGTAGGTACTCSequence (5' to 3,): TTCCTCCCCATGTCGTAGGTACTC

CTTAAAGTTAGTATTTTTATATGTCTTAAAGTTAGTATTTTTATATGT

AGTTTCTGAAGT 控制組DNA分子（/πΖ ssDNA片段） 合成序列長度：30 bpsAGTTTCTGAAGT control group DNA molecule (/πΖ ssDNA fragment) Synthetic sequence length: 30 bps

序列（5,至 3’）： TACGGCCAGGACAGTGCAACGGGCGSequence (5, to 3'): TACGGCCAGGACAGTGCAACGGGCG

CTGGG ii. 金奈米粒子探針之製備 於50μ140ηιη的金奈米粒子（〇.4Μο1/μ1)中加入10μ1 的5’端硫基化DNA引子（l〇〇〇pm〇l)，於室溫下反應16 小時後’加入 56μ1 的 0.1M PB buffer(pH 7.4)，再以 2M 的NaCl進行流洗作用’依序整反應液中NaCl之濃度為 0.05M、0.1M及0.2M，每次反應8小時，最後使該NaCl 201043701 濃度達至0.3M後離心’去除上清液後加入〇.3M NaCl 及1 OmM PB buffer(pH 7.0) ’混合後再離心，移除上清液 後，溶解於 500μ1 之 0.3M NaC卜 10mM PB buffer(pH 7.4) 及 0.01% azide(NaN3)之中。 iii·生物素DNA探針之購備 購自赛恩斯生物科技有限公司（此為商品化產品， 僅須提供DNA引子片段，即可購得所需之生物素〇ΝΑ 探針）。 〇 iv·目標DNA分子之檢測 如上述第一圖設計原理之流程，係於一長條型硝化 纖維膜（3.0公分χ〇.4公分）上固定抗-抗生物素蛋白抗 體(anti-avidin antibody) 15 pmol/ 卜 1 1 作為測試條 帶’再者，將待測DNA分子，目標DNA分子HPV : 1. 6 pmo卜 160fmo卜 16fmo卜 1.6fmol、160amol、16amol、 1.6amol、16〇zmol、16zmol、1.6zmol、0 ;及控制組 iac 〇 Z ssDNA : !〇 pmo卜金奈米粒子探針0.4 fmol/卜40 1及生物素DNA探針1 〇〇 pmol/ 1，1.2 1之反應液 升溫（至約95°C，2分鐘）’之後控制溫度（約45°C，2 0分鐘）’使該金奈米粒子探針及該生物素DNA探針與 該單股目標DNA分子進行互補黏合（其中該金奈米粒子 探針及該生物素DNA探針係分別與目標DNA分子之同 一侧單股互補），接著將反應後之反應液加入抗生物素蛋 白（avidin) 15 pmol/丨，2卜使該抗生物素蛋白與生物 素DNA探針結合’再將終反應加入已固定抗-抗生物素 11 201043701 蛋白抗體之硝化纖維膜上，其結果如第二圖所示。 於第二圖中，可明顯觀察到本發明中針對目標DNA 分子，可有效產生明顯呈色結果，不論目標濃度為多少f mol(l(T15mol)、amol(lCT18mol)或達至 16 zmol(l(T21mo 1)，本發明中用於债測DNA分子之金奈米粒子雜交層析 法都可以檢測出來，且空白試驗（水）及控制組(lacZ s sDNA)都不會被檢測出來，可見本發明方法具有一定之 專一性及靈敏度，可用於有效檢測目標DNA分子。 〇 實施例二：以lacZ ssDNA檢測對象 i.偵測lacZ ssDNA之DNA探針 金奈米粒子探針 引子長度：20 bps 引子序列（5,至 3，）： TTGAGCCCAGCGCCCGTTGC 生物素DNA探針 引子長度：20 bps |j. 引子序歹|](5,至 3，）： ACTGTCCTGGCCGTATTCTA-bio tin 目標DNA分子（lacZ ssDNA片段） 合成序列長度：30CTGGG ii. Preparation of the gold nanoparticle probe Add 10 μl of 5'-terminal thiolated DNA primer (l〇〇〇pm〇l) to 50μ140ηηη of gold nanoparticles (〇.4Μο1/μ1) at room temperature After 16 hours of reaction, '56 μl of 0.1 M PB buffer (pH 7.4) was added, followed by flow washing with 2 M NaCl. The concentration of NaCl in the reaction solution was 0.05 M, 0.1 M and 0.2 M, respectively. 8 hours, finally, the NaCl 201043701 concentration reached 0.3M, and then centrifuged 'removed the supernatant, then added 〇.3M NaCl and 1 OmM PB buffer (pH 7.0) 'mixed and centrifuged, remove the supernatant, dissolve in 500 μl of 0.3 M NaC in 10 mM PB buffer (pH 7.4) and 0.01% azide (NaN3). Iii· Purchase of biotin DNA probes purchased from Sainz Biotechnology Co., Ltd. (this is a commercial product, only the DNA primer fragment is required to obtain the desired biotin 探针 probe). 〇iv·Detection of target DNA molecules The flow of the design principle of the first figure above is based on a long strip of nitrocellulose membrane (3.0 cm χ〇.4 cm) immobilized anti-avidin antibody (anti-avidin antibody) 15 pmol / 卜 1 1 as a test strip 'again, the DNA molecule to be tested, the target DNA molecule HPV: 1. 6 pmo b 160fmo b 16fmo b 1.6fmol, 160amol, 16amol, 1.6amol, 16〇zmol, 16zmol , 1.6zmol, 0; and the control group iacon 〇Z ssDNA : !〇pmo 卜 纳米 nanoparticle probe 0.4 fmol / 卜 40 1 and biotin DNA probe 1 〇〇pmol / 1,1.2 1 reaction liquid temperature rise ( Up to about 95 ° C, 2 minutes) 'after controlling the temperature (about 45 ° C, 20 minutes)', the gold nanoparticle probe and the biotin DNA probe are complementaryly bonded to the single-stranded target DNA molecule ( Wherein the gold nanoparticle probe and the biotin DNA probe are complementary to the same side of the target DNA molecule, respectively, and then the reaction solution is added to avidin (avidin) 15 pmol/丨, 2 Binding the avidin to the biotin DNA probe' and then adding the final reaction to the immobilized anti- Biotin 11201043701 nitrocellulose membrane protein antibody, the second result as shown in FIG. In the second figure, it can be clearly observed that the target DNA molecule of the present invention can effectively produce a significant coloration result regardless of the target concentration of f mol(l(T15mol), amol(lCT18mol) or up to 16zmol(l). (T21mo 1), the hybridization chromatography of the gold nanoparticles used for the measurement of DNA molecules in the present invention can be detected, and the blank test (water) and the control group (lacZ s sDNA) are not detected, and it can be seen. The method of the invention has certain specificity and sensitivity, and can be used for effectively detecting target DNA molecules. 〇 Example 2: detecting a target with lacZ ssDNA i. detecting a DNA probe of lacZ ssDNA, a gold nanoparticle probe, introduction length: 20 bps Primer sequence (5, to 3,): TTGAGCCCAGCGCCCGTTGC Biotin DNA probe primer length: 20 bps | j. primer sequence 歹|] (5, to 3,): ACTGTCCTGGCCGTATTCTA-bio tin target DNA molecule (lacZ ssDNA fragment) synthesis Sequence length: 30

序列（5,至 3 ) : TACGGCCAGGACAGTGCAACGGGSequence (5, to 3) : TACGGCCAGGACAGTGCAACGGG

CGCTGGG 控制組DNA分子（hpv ssDNA片段） 合成序列長度：60 bps 12 201043701CGCTGGG control group DNA molecule (hpv ssDNA fragment) Synthetic sequence length: 60 bps 12 201043701

引子序列(5,至 3，）： TTCCTCCCCATGTCGTAGGTACT CCTTAAAGTTAGTATTTTTATAT GTAGTTTCTGAAGT ii.金奈米粒子探針之製備 於50μ1 40nm的金奈米粒子中加入10μ1的5’端硫 基化DNA引子（lOOOpmol)，於室溫下反應16小時後， 加入 56μ1 的 0.1M PB buffer(pH 7.4)，再以 2M 的 NaCl 進行流洗作用，依序整反應液中NaCl之濃度為0.05M、 〇 0.1M及0.2M，每次反應8小時，最後使該NaCl濃度達 至0.3M後離心，去除上清液後加入0.3M NaCl及10m Μ PB buffer(pH 7.0) ’混合後再離心，移除上清液後， 溶解於 500μ1 之 0.3M NaC卜 10mM PB buffer(pH 7.4) 及 0.01% azide(NaN3)之中。 iii·生物素DNA探針之購備Primer sequence (5, to 3,): TTCCTCCCCATGTCGTAGGTACT CCTTAAAGTTAGTATTTTTATAT GTAGTTTCTGAAGT ii. Preparation of the gold nanoparticle probe Add 10 μl of 5'-terminal thiolated DNA primer (100 pmol) to 50 μl of 40 nm gold nanoparticles at room temperature After 16 hours of reaction, 56 μl of 0.1 M PB buffer (pH 7.4) was added, followed by flow washing with 2 M NaCl, and the concentration of NaCl in the reaction solution was 0.05 M, 〇0.1 M and 0.2 M, respectively. After reacting for 8 hours, finally, the NaCl concentration was brought to 0.3 M, and then centrifuged. After removing the supernatant, 0.3 M NaCl and 10 m Μ PB buffer (pH 7.0) were added. After mixing, the mixture was centrifuged, and the supernatant was removed and dissolved in 500 μl. 0.3M NaC in 10 mM PB buffer (pH 7.4) and 0.01% azide (NaN3). Iii· Purchase of biotin DNA probe

購自賽恩斯生物科技有限公司（此為商品化產品， 〇 僅須提供DNA引子片段，即可購得所需之生物素DNA 探針）。 iv·目標DNA分子之檢測Purchased from Sainz Biotech Co., Ltd. (this is a commercial product, 〇 only need to provide DNA primer fragments, you can get the required biotin DNA probe). Iv·Detection of target DNA molecules

如上述第一圖設計原理之流程，係於一長條型石肖化 纖維膜上固定抗-抗生物素蛋白抗體(anti-avidin antibod y) 15 pmol/ 1 ’ 1 1作為測試條帶，再者，將待測DN A 分子，目標 DNA 分子 lacZ ssDNA : 160fmol、16fmol、 1.6fmo卜 160amo卜 16amo卜 1.6amol、160zmol、16zmol、 1.6zmol、0 ;及控制組HPV : 10 pmol、金奈米粒子探針 13 201043701 〇·4 fm0l/ 1，40 1 及生物素 dna 探針 10()pm〇1/ i 1.2 1之反應液升溫（至約95t，2分鐘），之後 溫度（約贼，2Q分鐘），使該金奈米粒子探針及^生 物素DNA探針與該單股目標職分子進行互補黏封其 中該金奈綠子騎及該生物素DNA探⑽分別與目 標DNA分子之同-側單股互補），接著將反應後之反應 液加入抗生物素蛋白（avidin)15 pm〇l/卜2卜使該抗 生物素蛋白與生物素DNA探針結合，再將終反應加二 固定抗-抗生物素蛋白抗體之硝化纖維膜上，其結果如第 三圖所示。 於第二圖中，可明顯觀察到本發明中針對目標DNA 分子(lacZ SSDNA) ’可有效產生明顯呈色結果，不論目 標濃度為多少fmol(10_15m〇i)，或者達至〗6 am〇1(1(rl8 mol) ’本發明中用於偵測DNA分子之金奈米粒子雜交層 析法都可以檢測出來’且空白試驗（水）及控制組(Hpv) 都不會被檢測出來，可見本發明方法具有一定之專一性 及靈敏度’可用於有效檢測目標DNA分子。 實施例三：以人類乳突病毒檢測對象（雙股DNA) i·偵測HVP之DNA探針 金奈米粒子探針 引子長度：35 bpsThe process of the design principle of the first figure above is to immobilize anti-avidin antibod y 15 pmol/ 1 ' 1 1 as a test strip on a long strip-shaped stone fiber membrane. DN A molecule to be tested, target DNA molecule lacZ ssDNA: 160fmol, 16fmol, 1.6fmo, 160amob, 16amo, 1.6amol, 160zmol, 16zmol, 1.6zmol, 0; and control group HPV: 10 pmol, gold nanoparticles Probe 13 201043701 〇·4 fm0l/ 1,40 1 and biotin dna probe 10 () pm 〇 1 / 1.2 1 1 temperature of the reaction solution (to about 95t, 2 minutes), then temperature (about thief, 2Q minutes And the complementary binding of the gold nanoparticle probe and the biotin DNA probe to the single-strand target molecule, wherein the Chennai green riding and the biotin DNA probe (10) are respectively the same as the target DNA molecule - The side single strands are complementary), and then the reaction mixture is added to avidin (avidin) 15 pm〇l/b 2 to bind the avidin to the biotin DNA probe, and then the final reaction is fixed. The result was shown in the third figure on the nitrocellulose membrane of the anti-avidin antibody. In the second figure, it can be clearly observed that the target DNA molecule (lacZ SSDNA) in the present invention can effectively produce a significant coloration result regardless of the target concentration of fmol (10_15 m〇i), or up to 〖6 am〇1 (1 (rl8 mol) 'The hybridization chromatography of the gold nanoparticles used for detecting DNA molecules in the present invention can be detected' and the blank test (water) and the control group (Hpv) are not detected. The method of the invention has certain specificity and sensitivity' can be used for effectively detecting target DNA molecules. Example 3: Detection of human papillomavirus (double-stranded DNA) i·Detecting HVP DNA probe, gold nanoparticle probe Lead length: 35 bps

引子序列（5,至 3，）： AAAAAAAAAAACTTCAGAAACPrimer sequence (5, to 3,): AAAAAAAAAAACTTCAGAAAC

TACATATAAAAATA 14 201043701 生物素DNA探針 引子長度：35 bpsTACATATAAAAATA 14 201043701 Biotin DNA Probe Primer Length: 35 bps

引子序列（5,至 3，）： biotin- AAAAAAAAAAGGAGTACCPrimer sequence (5, to 3,): biotin- AAAAAAAAAAGGAGTACC

TACGACATGGGGAGGAA 目標雙股DNA分子 以 BMY09 引子(AAAAGGATCCGCMCAGGGWCAT AAYAATGC)及 HMY11 引子(AGGCAAGCTTCGTCCMA RRGGAWACTGATC)當引子進行聚合酶鏈鎖反應，將Si 〇TACGACATGGGGAGGAA Target double-stranded DNA molecule The BMY09 primer (AAAAGGATCCGCMCAGGGWCAT AAYAATGC) and HMY11 primer (AGGCAAGCTTCGTCCMA RRGGAWACTGATC) were used as primers for polymerase chain reaction, and Si 〇

Ha細胞染色體上的晚期基因（LI region )構築至大腸桿 菌質體上，命名為pHC。純化質體後做測試，模擬在子 宮頸癌病變細胞中的target DNA。 控制組DNA分子（lacZ ssDNA片段） 合成序列長度：30 bpsThe late gene (LI region) on the chromosome of the Ha cell was constructed on the bacterium of Escherichia coli and designated as pHC. The plastids were purified and tested to mimic target DNA in cervical cancer cells. Control group DNA molecule (lacZ ssDNA fragment) Synthetic sequence length: 30 bps

引子序列（5,至 3’）： TACGGCCAGGACAGTGCAACGGPrimer sequence (5, to 3'): TACGGCCAGGACAGTGCAACGG

GCGCTGGG Ο ii·金奈米粒子探針之製備 於50μ1 40nm的金奈来粒子中加入10μ1的5’端硫 基化DNA引子（lOOOpmol)，於室溫下反應16小時後， 加入 56μ1 的 0.1M PB buffer(pH 7.4)，再以 2M 的 NaCl 進行流洗作用，依序整反應液中NaCl之濃度為0.05M、 0.1M及0.2M，每次反應8小時，最後使該NaCl濃度達 至0.3M後離心，去除上清液後加入0.3M NaCl及10m Μ PB buffer(pH 7.0)，混合後再離心，移除上清液後， 溶解於 500μ1 之 0.3M Naa、10mM PB buffer(pH 7.4) 15 201043701 及 0.01% azide(NaN3)之中。 iii·生物素DNA探針之購備 購自赛恩斯生物科技有限公司（此為商品化產品， 僅須提供DNA引子片段’即可購得所需之生物素DNa 探針）。 iv.目標DNA分子之檢測 如上述第一圖設計原理之流裎，係於一長條型確化 纖維膜（3.0公分χ〇.4公分）上固定抗_抗生物素蛋白抗 體(anti-avidin antibody) 15 pmol/ 1，ι 1 作為測試條 帶，再者，將待測DNA分子’目標DNA分子HPV : 24.8fmo卜 2.48fmo卜 248amo卜 24.8am〇卜 2.48amol、〇 ; 及控制組lacZ ssDNA : 10 pmol、金奈米粒子探針〇.4 fmol/ 1，4〇 1 及生物素 DNA 探針 100pm〇1/ b 丨 2 χ 之反應液升溫（至約95°C，2分鐘），以使其内之待測 DNA分子變性(denature)而打開雙股DNA分子成為單股 DNA分子’之後控制溫度（約45¾，20分鐘），使該金 奈米粒子探針及該生物素DNA探針與該單股目標dna 分子進行互補黏合（其中該金奈米粒子探針及該生物素 DNA探針係分別與目標DNA分子之同一侧單股互補）， 接著將反應後之反應液加入抗生物素蛋白（avidin) 15 pmol/ 1，2 1，使該抗生物素蛋白與生物素£)：^八探針 結合，再將終反應加入已固定抗·抗生物素蛋白抗體之硝 化纖維膜上，其結果如第四圖所示。 於第四圖中，可明顯觀察到本發明中針對目標DNa 16 201043701 分子’可有效產生明顯呈色結果，不論目標濃度為多少 fmol(l(T mol) ’ 或者達至 2 48 amol(l〇-18mol)，本發明中 用於彳貞測DNA分子之金奈米粒子雜交層析法都可以檢 測出來’且空白試驗（水）及控制組(lacZ SSDNA)都不會 被檢測出來，可見本發明方法具有一定之專一性及靈敏 度’可用於有效檢測目標DNA分子。 【圖式簡單說明】 第一圖為本發明一種用於偵測DNA分子之金奈米粒子 雜交層析法之示意圖。 第二圖為偵測目標DNA分子HPVssDNA之結果圖。 第三圖為偵測目標DNA分子lacZ ssDNA DNA之結果 圖。 第四圖為偵測雙股目標DNA分子HPV dsDNA之結果圖。 〇 【主要元件符號說明】 2 金奈米粒子探針 4 單股DNA分子 6 硝化纖維膜 8 吸收塾 1 待測DNA分子 3 生物素DNA探針 5 抗生物素蛋白 7 測試條帶 9 抗-抗生物素蛋白抗體 17Preparation of GCGCTGGG Ο ii·Genna nanoparticle probe Add 10 μl of 5'-terminal thiolated DNA primer (100 pmol) to 50 μl of 40 nm gold natrile particles, and react at room temperature for 16 hours, then add 56 μl of 0.1 M. PB buffer (pH 7.4), followed by 2M NaCl flow washing, the concentration of NaCl in the reaction solution was 0.05M, 0.1M and 0.2M, each reaction for 8 hours, and finally the NaCl concentration reached 0.3. After centrifugation, remove the supernatant, add 0.3 M NaCl and 10 m Μ PB buffer (pH 7.0), mix and centrifuge, remove the supernatant, and dissolve in 500 μl of 0.3 M Naa, 10 mM PB buffer (pH 7.4). 15 201043701 and 0.01% azide (NaN3). Iii· Purchase of biotin DNA probes purchased from Sainz Biotechnology Co., Ltd. (this is a commercial product, only the DNA primer fragment is required to obtain the desired biotin DNa probe). Iv. Detection of the target DNA molecule As described in the design of the first figure above, the anti-avidin antibody is immobilized on a long strip of deficient fiber membrane (3.0 cm 4.4 cm). 15 pmol / 1, ι 1 as a test strip, in addition, the DNA molecule to be tested 'target DNA molecule HPV: 24.8fmo 卜 2.48fmo 248amo 24.8am 〇 2.48amol, 〇; and control group lacZ ssDNA: 10 pmol, the gold nanoparticle probe 〇.4 fmol/ 1,4〇1 and the biotin DNA probe 100pm〇1/ b 丨2 χ The temperature of the reaction solution is raised (to about 95 ° C, 2 minutes), so that After the DNA molecule is denatured and the double-stranded DNA molecule is turned into a single-stranded DNA molecule, the temperature is controlled (about 453⁄4, 20 minutes), and the gold nanoparticle probe and the biotin DNA probe are The single-stranded target DNA molecule is complementarily bonded (wherein the gold nanoparticle probe and the biotin DNA probe system are respectively complementary to the same side of the target DNA molecule), and then the reaction solution after the reaction is added to the avidin The protein (avidin) 15 pmol / 1,2 1, makes the avidin and biotin £): ^ Eight probes were combined, and the final reaction was added to the nitrocellulose membrane to which the anti-avidin antibody was immobilized, and the results are shown in Fig. 4. In the fourth figure, it can be clearly observed that the target DNa 16 201043701 molecule can effectively produce significant coloration results regardless of the target concentration of fmol (l(T mol) ' or up to 2 48 amol (l〇 -18 mol), the hybridization chromatography of the gold nanoparticles used for speculating DNA molecules in the present invention can be detected' and the blank test (water) and the control group (lacZ SSDNA) are not detected. The method of the invention has certain specificity and sensitivity' can be used for effectively detecting target DNA molecules. [Simplified illustration] The first figure is a schematic diagram of a hybridization chromatography method for detecting gold nanoparticles of DNA molecules. The second picture is the result of detecting the target DNA molecule HPVssDNA. The third picture is the result of detecting the target DNA molecule lacZ ssDNA DNA. The fourth picture is the result of detecting the double-strand target DNA molecule HPV dsDNA. DESCRIPTION OF SYMBOLS 2 Gold Nanoparticle probe 4 Single strand DNA molecule 6 Nitrocellulose membrane 8 Absorption 塾1 DNA molecule to be tested 3 Biotin DNA probe 5 Avidin 7 Test strip 9 Anti-Avidin antibody 17

Claims (1)

201043701 七、申請專利範圍： 1. 一種用於偵測目標DNA之奈米金粒子雜交層析法，其係包 含下列步驟： ~ a. 將抗抗生物素蛋白抗體（anti_avidjn antib〇dy)固定於石肖化 纖維膜上； ' b. 將待測DNA、金奈米粒子探針及生物素DNA探針混合， 並升高溫度（用於打開待測DNA之雙股，約9〇°c，2分 鐘）後控制溫度（約45¾，20分鐘）’使該等探針與單股 DNA進行互補黏合（其中該金奈米粒子探針及該生物素 DNA探針係分別與目標同一側單股互補）； c. 將步驟b之反應液加入抗生物素蛋白（avidin)，使其與生物 素DNA探針結合； d. 將步驟c反應液流經步驟a之硝化纖維膜以進行抗_抗生物 素蛋白抗體針對抗生物素蛋白之捕捉，若有呈色（紅色） 反應，則待測DNA中含有目標DNA。 2. 如申請專利範圍第1項之奈米金粒子雜交層析法，其中該目 標DNA為人類乳突瘤病毒DNA之片段。 3·如申請專利範圍第2項之奈米金粒子雜交層析法，其中該待 測DNA係為經純化之DNA分子。 4. 如申請專利範圍第3項之奈米金粒子雜交層析法，其中該目 標DNA分子之單股片段序列為（5’至3，）： TTCCTCCCCATGTCGTAGGTACTCCTTAAAGTTAGTATT TTTATATGTAGTTTCTGAAGT。 5. 如申請專利範圍第1項之奈米金粒子雜交層析法，其中該待 201043701 測DNA之债測靈敏度可達amol(10·18 mol)。 6.如申請專利範圍第1項之奈米金粒子雜交層析法，其中該抗 -抗生物素蛋白抗體（anti-avidin antibody)之濃度為15 pmol/ 1 ;該金奈米粒子探針濃度為0.4 fmol/ 1 ;該生物素 DNA探針濃度為lOOpmol/ 1;該抗生物素蛋白（avidin)之濃 度為 15 pmol/ 1。201043701 VII. Patent application scope: 1. A nanogold hybridization chromatography method for detecting target DNA, which comprises the following steps: ~ a. immobilizing anti-avidin antibody (anti_avidjn antib〇dy) on Stone on the fiber membrane; ' b. Mix the DNA to be tested, the gold nanoparticle probe and the biotin DNA probe, and raise the temperature (used to open the double strand of the DNA to be tested, about 9 ° ° C, 2 minutes) control temperature (about 453⁄4, 20 minutes)' to make the probes and the single-strand DNA complementary binding (where the gold nanoparticle probe and the biotin DNA probe system are respectively on the same side as the target Complementary); c. adding the reaction solution of step b to avidin to bind to the biotin DNA probe; d. flowing the reaction solution of step c through the nitrocellulose membrane of step a for anti-resistant The biotin antibody is directed against the capture of avidin. If there is a color (red) reaction, the DNA to be tested contains the target DNA. 2. Nanoparticle hybridization chromatography as claimed in claim 1 wherein the target DNA is a fragment of human papillomavirus DNA. 3. Nanoparticle hybridization chromatography as claimed in claim 2, wherein the DNA to be tested is a purified DNA molecule. 4. The nanogold particle hybridization chromatography according to item 3 of the patent application, wherein the single-strand fragment sequence of the target DNA molecule is (5' to 3,): TTCCTCCCCATGTCGTAGGTACTCCTTAAAGTTAGTATT TTTATATGTAGTTTCTGAAGT. 5. For the nanogold particle hybridization chromatography method of claim 1, the sensitivity of the test for 201043701 DNA can reach amol (10·18 mol). 6. The nanogold particle hybridization chromatography according to claim 1, wherein the concentration of the anti-avidin antibody is 15 pmol/l; the concentration of the gold nanoparticle probe The concentration was 0.4 fmol/1; the concentration of the biotin DNA probe was 100 pmol/l; the concentration of the avidin was 15 pmol/l. 1919