【摘要】：癌癥是危害人類健康的三大疾病之一,對腫瘤標(biāo)志物的監(jiān)測在疾病診斷和治療中起重要作用,早診早治能有效的預(yù)防并延緩腫瘤的發(fā)生與發(fā)展,降低癌癥死亡率。而傳統(tǒng)的免疫分析多在界面上進(jìn)行,需要多次的分離、沖洗操作,造價(jià)高,耗時(shí)長,不適用于大規(guī)模篩查檢測。鄰位誘導(dǎo)策略是通過蛋白識別誘發(fā)DNA鄰位組裝,最終將對大分子蛋白質(zhì)的檢測轉(zhuǎn)換為對核酸的檢測,通過與各種核酸放大策略相結(jié)合,可獲得高的檢測靈敏度。本論文基于鄰位誘導(dǎo)策略,結(jié)合核酸酶切放大,以建立簡單、均相、靈敏的免疫分析方法為目標(biāo),開展了以下工作:提出了一種基于靶標(biāo)誘導(dǎo)G-四鏈體/hemin DNA酶循環(huán)生成的比色及化學(xué)發(fā)光成像免疫分析策略,用于簡單、均相的腫瘤標(biāo)志物的檢測。所設(shè)計(jì)的體系中,以兩個(gè)DNA-抗體偶聯(lián)物(Ab1-DNA1,Ab2-DNA2)為識別單元,在靶標(biāo)蛋白存在時(shí),通過免疫識別,形成Ab1-DNA1/Target/Ab2-DNA2鄰位復(fù)合物,在該鄰位復(fù)合物中,由于鄰位效應(yīng),DNA1與DNA2進(jìn)行雜交,進(jìn)而鏈取代雙鏈DNA(DNA3/DNA4)上的DNA3,并釋放出富含G堿基的DNA4。DNA4與hemin結(jié)合后,在位形成G-四鏈體/hemin DNA酶,進(jìn)一步利用核酸外切酶Ⅲ剪切DNA3,而釋放鄰位復(fù)合物,使得鏈取代反應(yīng)循環(huán)進(jìn)行,在位生成大量的G-四鏈體/hemin DNA酶。該DNA酶可以催化3,3',5,5'-四甲基聯(lián)苯胺氧化其顏色從無色變?yōu)樗{(lán)色,或催化魯米諾-H_2O_2產(chǎn)生化學(xué)發(fā)光。產(chǎn)生的信號可直接裸眼,或利用比色和化學(xué)發(fā)光成像的方法讀出。以癌胚抗原CEA為靶標(biāo)蛋白,使用比色法和化學(xué)發(fā)光成像法進(jìn)行分析,檢測范圍都可達(dá)4個(gè)數(shù)量級,檢測限分別為170和16pg/mL。該方法操作簡便、檢測靈敏度高、目標(biāo)靈活、且信號讀出方式多樣,具有良好的商業(yè)應(yīng)用前景。
[Abstract]:Cancer is one of the three diseases harmful to human health. The monitoring of tumor markers plays an important role in the diagnosis and treatment of diseases. Early diagnosis and early treatment can effectively prevent and delay the occurrence and development of tumors and reduce cancer mortality. However, the traditional immunoassay is mostly carried out on the interface, which requires many times of separation, washing operation, high cost and time consuming, so it is not suitable for large-scale screening and testing. The strategy of site-inducing is to induce the assembly of DNA sites by protein recognition, and to convert the detection of macromolecular proteins into nucleic acid detection. By combining with various nucleic acid amplification strategies, high detection sensitivity can be obtained. The aim of this paper is to establish a simple, homogeneous and sensitive immunoassay based on the strategy of orthotopic induction and nucleic acid digestion amplification. The following works are carried out: a colorimetric and chemiluminescence imaging immunoassay strategy based on target induced G-quad / hemin DNA enzyme cycle is proposed for simple and homogeneous detection of tumor markers. In the designed system, two DNA- antibody conjugates (Ab1-DNA1,Ab2-DNA2) were used as recognition units. In the presence of the target protein, the Ab1-DNA1/Target/Ab2-DNA2 neighbor complex was formed by immunological recognition. In the adjacent complex, DNA1 hybridized with DNA2 because of the orthotopic effect. Then the DNA3, on the double-stranded DNA (DNA3/DNA4) was replaced by the chain and the G-rich DNA4.DNA4 was released to bind with hemin, and then the G-quadruplex / hemin DNA enzyme was formed in situ. Furthermore, the nucleic acid exonuclease 鈪，

本文編號：2290826