自增强型金属有机框架纳米信号探针用于miRNA超敏无酶ECL检测的实验研究

Studies have shown that the circulating miRNA become a new biomarker for the early diagnosis, classification and prognosis of malignant tumors. However, it lacks of convenient and sensitive testing methods due to fewer basic researches on the new detection technologies. With its high sensitivity, specificity, space-time controllability, etc., the novel electrochemiluminescence nucleic acid sensing technology will make up for the deficiency of the existing miRNA detection method. Fortunately, our previous study achieved the efficient and specific recognition of target miRNA through the multi-coordinated nano-sensing interface constructed by self-assembling hairpin-type locked nucleic acids in the thiol-hexanol matrix. On this basis, the project was designed to establish a thermostatical and enzyme-free target catalyzed hairpin assembly technique for the circulating miRNA in the serum of patients with primary hepatic carcinoma. Two kinds of luminophores were fixed in the metal-organic framework (MOFs) using the solvothermal and chemical cross-linking method, and the signal probes were labeled with “click” chemical method on the outer of luminescence-functionalized MOFs, to construct the self-enhanced nano-signal probes nanocomposites. And then, a novel electrochemiluminescence nucleic acid sensor was prepared. Thus, the electrochemical response pattern of signal cascade amplification with higher sensitivity and efficiency was formed on the micro/nano interface. The high-throughput detection will be further realized via the screen printed electrode array assay. Ultimately, a sensitive, specific and enzyme-free detection of miRNA was established, which provides an innovative technology platform for the diagnosis of malignant liver neoplasms.

循环miRNA现已成为恶性肿瘤早期诊断、分型和预后研究的一种新兴标志物，但受限于检测技术基础研究的不足，临床仍然缺乏便捷、灵敏的检验手段。而新型电致化学发光核酸传感技术以其高敏特异、时空可控等特点，有望弥补现有miRNA检验技术的不足。课题组前期已通过巯基己醇矩阵中自组装发夹型锁核酸构筑的多元协调传感界面，实现了对靶分子miRNA的高效特异识别。本课题拟在此基础上，以原发性肝癌血清中循环miRNA为靶标，建立恒温无酶高效的催化发夹组装技术；运用溶剂热法、化学交联法将两种发光体固载在金属有机框架中，采用“click”化学法标记信号探针，构筑自增强纳米信号探针；制备新型的电致化学发光核酸传感器。从而在微纳米界面上，形成灵敏度和效率更高的信号级联放大响应模式，并进一步通过丝网印刷电极阵列技术实现高通量检测，建立对miRNA灵敏特异、无酶高效的检测新技术，为恶性肿瘤的诊断提供一种创新性的技术平台。

循环miRNAs作为无创生物标志物被广泛研究，其能够提高疾病早期诊断和预后判断的准确度。但受限于检测技术基础研究的不足，临床仍缺乏便捷、灵敏的检验手段。基于此本项目展开基于分子信标样催化发夹组装(mCHA)与双重纳米酶反应的多重信号放大的检测技术研究。本项目创新性引入信号转导探针作为微小RNA的识别和转导元件，构建了mCHA DNA分子电路，实现靶分子的精准识别和无酶、高效循环；构筑了双纳米酶增强型金属有机框架纳米信号探针，实现信号级联放大；构建了多元协调的、稳定性好的传感微纳米界面，调控捕获探针导向和降低杂质吸附和机制干扰。基于此，建立了一种通用性核酸检测策略和平台，特异性强、检测限低至0.25 fM，并成功应用于循环miR-721的检测，有望为疾病的早诊断、药物干预以及疾病进展、治疗效用的实时评估提供了一种新的检测平台，加速miRNAs检测在临床中的应用。本课题历时3年，共发表标注有基金号的文章3篇，其中SCI论文2篇，IF>10分1篇。本项目的顺利实施为后续基于纳米材料的生物应用研究奠定了坚实的理论和实验基础。