基于纳米电极表面离子开关与电化学催化协同效应的miRNA检测方法研究

MicroRNAs (miRNA) are short (～22 nucleotides) non-coding RNA molecules that regulate gene expression. Since their dysregulation leads to development of cancers, miRNAs have recently been proposed as being useful in diagnostics as biomarkers. Unfortunately,their characteristics like low abundance ,high degree of sequence similarity and extremely small size of mature miRNAs make it challenging to analysis miRNA with high sensitivity and specificity. Recently, nanoelectrochemical miRNA biosensors attract a lot of research interest for their advantages of sensitivity, small amount of starting sample and label-free analysis. In this project, we firstly assemble the PNA detection probes onto the surface of a nano-tip electrode. With the hybridization of target miRNAs, the probe will turn their configuration from free soft single-strand to rigid double-strand, which will construct a special "ion-gating" effect of [Fe(CN)6]3- on the surface of the nanotip electrode. In the meantime, the adsorption of [Ru(NH3)6]3+ will be increased for the increased negative charge on the electrode surface. As a result, the electrocatalysis signal of RuIII/FeIII will be amplified. This method is constructed on a nano-sized electrode, which need less amount of starting materials. We combined the "ion gating" effect with the electrocatalysis to improve the sensitivity. Based on the systemic optimization of the detection probes and their assembling on the electrode surface, the nonspecific absorption will be avoided. In all, We will aim at a extremely sensitive, specific and label free miRNA analysis method which will lay a good foundation for the future application in diagnostics. We will systematically investigate all the key reaction conditions with the advanced characterization technologies (SEM, TEM, XPS etc.), electrochemical analysis and traditional miRNA analysis methods (RT-PCT, digital PCR). By expatiating the mechanism, this project will provide valuable references for development of other nano-material biosensors.

微小RNA（miRNA）具有调控基因表达的作用，是潜在肿瘤分子靶标，但其分子短、丰度低、序列高度相似，给检测方法提出挑战。本项目提出基于纳米针尖电极表面离子开关与电化学催化协同效应，构建新型miRNA检测方法。在纳米金电极表面组装肽核酸（PNA）探针，特异性识别目标miRNA；探针与miRNA杂交后从柔性松散单链变成刚性直立双链，在电极表面形成铁氰离子扩散开关效应；且电极表面的miRNA可增加六氨合钌离子吸附量，提高催化电化学信号，实现目标miRNA检测。本项目将"离子开关"效应与电化学催化结合，提高检测灵敏度；采用纳米尺度电极，降低了样品需求量；改进表面探针，降低非特异吸附，实现高灵敏度、高特异性、无标记miRNA检测。我们将结合先进表征技术（STM，TEM，XPS等），综合考察各反应条件，阐述机理，形成其他纳米生物传感研究可借鉴的理论模型，为临床诊断应用奠定基础。

微小RNA（miRNA）具有调控基因表达的作用，是潜在肿瘤分子靶标，但其分子短、丰度低、序列高度相似，给检测方法提出挑战。本项目一个主要研究课题就是基于纳米针尖电极表面离子开关与电化学催化协同效应，构建新型miRNA 检测方法。对于电化学杂交传感器来说，如何提高杂交反应发生前后的信号变化量是一个重要的技术关键。研究中，我们在纳米金电极表面组装捕获探针，特异性识别目标miRNA，探针与miRNA杂交后从柔性松散单链变成刚性直立双链，在电极表面形成离子扩散开关效应：当纳米电极表面仅组装有单链DNA探针的时候，Fe(CN)63-可以比较容易的扩散到电极表面，发生电化学氧化还原反应，并产生一个明显的循环伏安（CV）特征峰（2.5V），而此时由于Ru(NH)63+浓度较低，其CV峰很小；当加入目标miRNA与探针杂交之后，表面密度显著增加，阻碍了离子扩散，促使Fe(CN)63-优先与吸附于探针表面的Ru(NH)63+发生催化反应，Ru(NH)63+的氧化还原特征峰增加，而Fe(CN)63-的特征峰变得很小，几乎消失。经过DPV定量分析，杂交前后，信噪比达到超过1000倍。首次实现了在单个纳米材料单元表面进行DNA杂交反应开关的电化学催化。本方法具有便携化、植入式检测潜能，未来具有个性化诊疗应用的良好前景。为了进一步提高miRNA检测灵敏度，我们发展了一种基于DNA纳米结构的电化学miRNA生物传感器的检测方法。通过采用三维的DNA纳米结构界面修饰方法，我们发现检测灵敏度可达埃摩尔水平（<1000 拷贝），并且具有很好的单碱基识别能力。该方法良好的重复性、不需要进行标记和无需PCR扩增等优势对方便、可靠的检测和分析食管鳞状细胞癌病人临床样品提供了有力保障。所研究的DNA纳米界面技术具有非常好的普适性。被成功应用于茎环结构探针miRNA传感器（E-DNA）研发以及端粒酶活性检测当中。