基于上转换荧光纳米粒子表面核酸等温延伸microRNA信息检测技术研究

Increasing evidence indicates that aberrant microRNA(miRNA) expression is a common feature of human diseases including cancers, cardiovascular disorders, metabolic disturbances and other serious diseases. These biomolecules have great potential for the early diagnosis of human diseases. However, several unique characteristics of miRNAs, including their small size, sequence homology among family members, and low abundance in total RNA samples, make it difficult to analyze the miRNA. Accordingly, the current technologies to detect miRNA have many drawbacks, such as low sensitivity and specificity, expensivity, and time-exaustivity. Herein, we propose a high sensitive and specific, and simple-executive miRNA imformation obtaining technique based on isothermal extension of nuclear acids chain on the surface of NaYF4:Yb,Er upconversion nanoparticles. An exponential extension process is only happened efficiently in the presence of target miRNA which provides additional stability for the universal primer on the surface of upconversion nanoparticles and specific primer in the solution. Associated by the DNA polymerase, both universal primer and specific primer will be extented and Cy3-labeled dUTP will be inserted. Thus, an effective fluorescence resonance energy transfer(FRET) system will be constructed. In this FRET system, NaYF4:Yb,Er upconversion nanoparticles as energy donor transfer energy to Cy3 molicules. Therefore, greatly enhanced intensity of emission fluorescence at 570 nm will be checked under excitating with 980 nm light, and we can detect the miRNAs by measuring the emission fluorescence intensity at 570 nm. This new method should provide promising diagnostic approaches for highly sensitive and specific, and simple-executive detection of miRNAs.

研究表明microRNA(miRNA)在癌症、心血管等重大疾病中表达异常，获得这些特异性miRNA表达信息可为疾病早期诊断提供重要依据。但由于miRNA自身特点，其检测较难。目前检测方法存在特异性不高、费用昂贵、操作复杂等缺点。本项目结合碱基堆积原理、等温延伸技术以及荧光共振能量转移（FRET）原理，建立新型的miRNA信息获取平台。该平台利用目标miRNA提供的碱基堆积力帮助特异性引物与连接在NaYF4:Yb,Er荧光纳米粒子表面的通用引物互补配对，并进行等温延伸。在延伸的过程中，引入Cy3标记的dUTP。从而以荧光纳米粒子为能量供体，Cy3为受体，构建FRET体系。无目标miRNA存在时，在操作温度下特异引物与通用引物不能结合，因而不能发生延伸。本方法无需反转，操作简单，且通过FRET可有效降低背景干扰达到高灵敏检测的目的，适合早期诊断与临床检测。

稀土上转换荧光纳米颗粒（Rare earth upconversion fluorescence nanoparticles, UCNPs）是近年来备受瞩目的重要发光材料。这种荧光纳米材料不仅发光强度高、光稳定性好，而且因其激发光为红外光，可有效避免生物样品自发荧光干扰，从而降低检测背景，提高生物检测的灵敏度。本项目主要是以稀土氯化物为原料，开发其在生物诊断方面的应用，主要内容包括以下三个方面：1利用水热法以及热分解法制备了NaYF4:Yb, Er 上转化粒子，并通过不断优化摸索出最佳的制备条件；2 采用变性牛血清蛋白来修饰油酸包覆的NaYF4: Yb, Er上转换纳米颗粒，并将其与氨基化探针进行酰胺反应，形成探针修饰的NaYF4: Yb, Er上转换纳米颗粒；3 利用基于NaYF4:Yb, Er上转化粒子的特殊性以及碱基堆积理论构建了一个新型无需扩增的检测miRNA的平台，并以NaYF4 :Yb, Er荧光强度为内参来定量检测目标DNA或miRNA的浓度。由于该方法无需反转录也无需进行扩增反应，且利用UCNP所发出的荧光作为内参可很大程度的减少系统误差，因此是一种理想的具有潜在应用价值的检测方法。此外,在项目的进行中，我们也制备和修饰了一系列的纳米材料。通过修饰这些纳米粒子表面拥有很多功能基团，具有良好的生物相容性。在本项目的资助下，我们共发表论文12篇，其中SCI 论文10篇，北大中文核心期刊以及EI各一篇，其中作为第一作者或通讯作者发表SCI论文9篇，最高影响因子为9.407（Chemistry of Materials，2014.3.11，26(5)：1794~1798）。授权专利1项，实审1项，待申请一项。到目前为止，共同培养研究生3名。