基于金属纳簇与G-四链体的多种miRNA序列的高通量电化学检测

MiRNA is closely related to the evolution and progression of various cancers, and therefore serves as an efficient biomarker in cancer diagnosis, treatment, and curative effects monitoring. As a result, development of highly sensitive, selective and high-throughput methods for the determination of various miRNA sequences is of great biological significance to early warning and diagnosis of cancers. In this proposal, relying on the properties of the DNA sequence specificity in metal nanoclusters and G-quadruplex synthesis and the high metal atom/ion content, high-throughput electrochemical assay of various miRNA sequences from sera of cancer patients has been carried out on one electrode. The sensitivity of the method can be further improved via loading a large number of metal nanoclusters and G-quadruplexes on the surface of carbon nanotubes. Various factors, such as the effective and controlled immobilization of the DNA probe, elimination of nonspecific adsorption, effect of metal nanoclusters and G-quadruplex synthesis on the hybridization reaction and the subsequent electrochemical assay, and improvement of the biosensor selectivity have been examined. The proposed method will circumvent the problems such as complicated procedures and sample pretreatment involved in polymerase chain reaction (PCR), photobleaching and less stability inherent in fluorescent dyes, low sensitivity and large RNA consumption in Northern Blotting. The sensing protocol will provide new insight and accurate information on the clinical diagnosis and treatment of glioma and other cancer patients.

MiRNA与肿瘤发生、发展密切相关，是肿瘤诊断、治疗与疗效监测过程中有效的生物标志物。开发高灵敏、高通量、高选择性检测多种miRNA序列的方法对癌症的预警及早期诊断具有重要的生物学意义。本项目基于金属纳簇与G-四链体形成时的碱基序列特异性以及富含金属原子/离子的特性，在同一电极表面建立了高通量检测肿瘤患者血清中多种miRNA靶序列的电化学方法。通过碳纳米管上负载大量的金属纳簇与G-四链体以提升方法的灵敏度。考察了DNA探针的有效可控固定、非特异性吸附的消除、金属纳簇与G-四链体的形成对杂交反应与电化学响应的影响、以及提高传感器的选择性等性能指标。该方法避免了聚合酶链式反应（PCR）操作步骤繁琐且涉及样品预处理、荧光染料易发生光漂白和稳定性较差以及Northern 印迹法敏感度低、RNA用量大等一系列问题。所开发的方法将为胶质瘤及其它癌症患者的临床诊断及治疗提供新的思路及可靠信息。

MiRNA与肿瘤发生、发展密切相关，是肿瘤诊断、治疗与疗效监测过程中有效的生物标志物。开发高灵敏、高选择性检测多种miRNA序列的方法对癌症的预警及早期诊断具有重要的生物学意义。采用磁球-纳米金复合物负载发卡结构的DNA探针以及二茂铁覆盖的纳米金/亲和素复合物，实现了胶质瘤患者血清中miRNA-182的高灵敏测定，miRNA-182的检出限为0.14 fM，且胶质瘤患者血清中miRNA含量约为正常人的2.9倍。该方法灵敏度高、选择性好，对磁球-纳米金复合物的磁分离和磁收集避免了实际样品测定中共存组分的干扰。利用磁球-纳米金复合物和嵌段DNA-纳米金复合物结合同时检测了胶质瘤患者血清中miRNA-182和miRNA-381序列，由于嵌段DNA-纳米金复合物表面可覆盖大量的二茂铁与亚甲基蓝基团，所以电化学信号被显著放大。miRNA-182和miRNA-381的检测限可达到0.20 fM 和0.12 fM。结果表明，I-II级、III-IV级胶质瘤病人血清中miRNA-182的表达水平分别是正常人血清的2-3倍与3-4倍，而miRNA-381的表达水平分别是正常人血清的2-3倍与4-5倍。采用悬挂3′-CCC 和CCT-5′序列的分子信标合成银纳米簇，基于银纳米簇的荧光特性变化，实现了方便、快捷的miRNA靶序列的检测。另外，以富含胞嘧啶碱基的DNA为模板合成了银纳米簇，利用银纳米簇的荧光特性实现了蛋白激酶活性的检测及酶抑制剂的筛选。为提升银纳米簇的光化学性能，采用哑铃状结构的金属硫蛋白为模板合成了量子产率高、荧光强度大、生物相容性好的银纳米簇，并实现了细胞成像以及miRNA等生物标志物的检测。通过在银纳米粒子表面合成银纳米簇制备了单金属纳米杂合体，实现了对银纳米簇稳定性及荧光的增强。所开发的方法避免了聚合酶链式反应操作步骤繁琐且涉及样品预处理、荧光染料易发生光漂白和稳定性较差以及Northern 印迹法敏感度低、RNA用量大等一系列问题，将为胶质瘤及其它癌症患者的临床诊断及治疗提供新的思路及可靠信息。