基于金刚石纳米阵列的小分子核糖核酸检测技术及其在高通量原位分析神经细胞异质性的应用

Intracellular miRNAs play critical roles in many physiologic and pathogenic activities. It has been reported that miRNAs are closed related to complex brain function and many neurodegenerative diseases. In this project, we aim to develop a highly sensitive non-PCR technique for fast, cheap, in-situ isolation and analysis of intracellular miRNAs from live cells. This technique is based on a “molecular fishing” system, which utilizes an array of diamond nanoneedles as minimum-invasive sensing units to access the intracellular space without hurting the cells. The interfacing between live cells and the diamond nanostructure will be delicately controlled by a centrifugation process, which enables large-scale delivery of probe molecules into live cells and also isolation of biomarkers out of them. Specifically in our implementation, upon a poke of cell membrane by the diamond nanoneedles, the specially designed probes (complimentary DNA for specific miRNAs) will be delivered into cytoplasm for binding with target miRNAs, and subsequently be captured by a general miRNA-binding-protein that has been crosslinked to the nanoneedle surface. The retrieval of the nanoneedle array would effectively perform the miRNA isolation. And then, a simple isothermal amplification procedure that is based on hybridization chain reaction（HCR）will be applied to the nanoneedle complexes (nanoneedles attached with protein-miRNA) for signal amplification to significantly boost the detection sensitivity to the level comparable to qRT-PCR analysis. In combination with machine learning and bioinformatics approaches, we will use this technique to analyze the cellular heterogeneity of neural stem cell different ion based on miRNA profiling, and also to study the change of miRNA expression spectrum in the hippocampal tissues, when animal brain undergoes memory, learning, injury and regeneration activities.

小分子核糖核酸（miRNA）在许多生理和病理活动中都起着非常重要的作用。研究表明，miRNA与多种神经退化疾病、神经异质性及复杂大脑功能有密切联系。但是，快速、高通量、多通道的在体分析miRNA的技术手段仍然十分有限。本项目拟研发一项基于金刚石纳米结构阵列的，以非PCR的原理，从活体细胞和组织内分离检测miRNA的方法。该方法利用精确可控的微创手段将基于金刚石的纳米结构和特异的核酸分子探针导入细胞内部，通过铰链在纳米结构表面的广谱miRNA结合蛋白实现快速且特异的从细胞质内分离一系列目标miRNA的目的，并进一步利用恒温链式杂交反应进扩增分离的miRNA分子以极大的提高其分析的灵敏度。利用该方法，结合人工智能和生物信息技术，我们将对神经干细胞的分化过程中产生的不同细胞miRNA表达谱的异质性及大脑海马区域神经组织在学习、记忆、创伤及修复等活动中的miRNA的表达谱变化做深入细致的研究。

小分子核糖核酸（miRNA）在许多生理和病理活动中都起着非常重要的作用。研究表明，m iRNA与多种神经退化疾病、神经异质性及复杂大脑功能有密切联系。但是，快速、高通量、多通道的在体分析miRNA的技术手段仍然十分有限。本项目研发一项基于金刚石纳米结构阵列的，以非PCR的原理，从活体细胞和组织内分离检测miRNA的方法。该方法利用精确可控的微创手段将基于金刚石的纳米结构和特异的核酸分子探针导入细胞内部，通过铰链在纳米结构表面的广谱miRNA结合蛋白实现快速且特异的从细胞质内分离一系列目标miRNA的目的，并进一步利用恒温链式杂交反应进扩增分离的miRNA分子以极大的提高其分析的灵敏度。利用该方法，结合人工智能和生物信息技术，我们对神经干细胞的分化过程中产生的不同细胞类型的miRNA表达谱的异质性进行了深入研究，并在健康和神经分裂症小鼠模型中研究大脑海马区域神经组织在学习、记忆等活动中的miRNA的表达谱演化，探究相关时间和空间上的异质性关联。

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