靶标诱导DNA酶循环生成的均相免疫分析
发布时间：2018-10-24 08:12
【摘要】：癌症是危害人类健康的三大疾病之一,对肿瘤标志物的监测在疾病诊断和治疗中起重要作用,早诊早治能有效的预防并延缓肿瘤的发生与发展,降低癌症死亡率。而传统的免疫分析多在界面上进行,需要多次的分离、冲洗操作,造价高,耗时长,不适用于大规模筛查检测。邻位诱导策略是通过蛋白识别诱发DNA邻位组装,最终将对大分子蛋白质的检测转换为对核酸的检测,通过与各种核酸放大策略相结合,可获得高的检测灵敏度。本论文基于邻位诱导策略,结合核酸酶切放大,以建立简单、均相、灵敏的免疫分析方法为目标,开展了以下工作:提出了一种基于靶标诱导G-四链体/hemin DNA酶循环生成的比色及化学发光成像免疫分析策略,用于简单、均相的肿瘤标志物的检测。所设计的体系中,以两个DNA-抗体偶联物(Ab1-DNA1,Ab2-DNA2)为识别单元,在靶标蛋白存在时,通过免疫识别,形成Ab1-DNA1/Target/Ab2-DNA2邻位复合物,在该邻位复合物中,由于邻位效应,DNA1与DNA2进行杂交,进而链取代双链DNA(DNA3/DNA4)上的DNA3,并释放出富含G碱基的DNA4。DNA4与hemin结合后,在位形成G-四链体/hemin DNA酶,进一步利用核酸外切酶Ⅲ剪切DNA3,而释放邻位复合物,使得链取代反应循环进行,在位生成大量的G-四链体/hemin DNA酶。该DNA酶可以催化3,3',5,5'-四甲基联苯胺氧化其颜色从无色变为蓝色,或催化鲁米诺-H_2O_2产生化学发光。产生的信号可直接裸眼,或利用比色和化学发光成像的方法读出。以癌胚抗原CEA为靶标蛋白,使用比色法和化学发光成像法进行分析,检测范围都可达4个数量级,检测限分别为170和16pg/mL。该方法操作简便、检测灵敏度高、目标灵活、且信号读出方式多样,具有良好的商业应用前景。
[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，
本文编号：2290826
[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，
本文编号：2290826
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