基于叠加内标策略和酶切放大技术的BRCA1基因电化学传感研究

It is of great importance to detect specific sequences on breast cancer susceptibility gene 1 (BRCA1) for early-stage diagnosis and treatment of breast cancer. All of the reported electrochemical sensors for BRCA1 gene analysis are based on single-signaling strategy, which is not beneficial for eliminating adverse effects on data reproducibility caused by factors such as electrode areas, DNA probes loading densities, complex components in real samples, instruments and so on. This seriously constrains the development of electrochemical sensors for BRCA1 gene analysis. Although the internal standard sensing strategy is helpful to solve the above problem, the electrochemical DNA sensors with internal control reported in literature needs technically difficult labeling strategy, which limits the application of internal control strategy in BRCA1 analysis. What's more, it could not meet the sensitivity requirement of BRCA1 gene detection in complex systems by only using internal control strategy. Based on the above issues, this project intends to develop a highly sensitive electrochemical sensor for BRCA1 gene detection by combining internal control, signal superposition strategy and exonuclease assisted signal amplification. The new sensing strategy can not only greatly improve the repeatability of electrochemical sensors, but also greatly reduce the detection limit.

对BRCA1基因序列的分析在促进乳腺癌的早期临床诊断和治疗方面具有深远意义。已报道的BRCA1基因电化学传感器都是基于单信号响应策略建立起来的，不利于消除电极面积、DNA探针固定密度、实际样品中的复杂成分及设备等因素对数据重复性的不良影响，严重制约了BRCA1基因电化学传感器的发展。虽然内标策略能改善复杂样品中BRCA1基因检测结果重复性，但已报道的内标型电化学DNA传感器对标记技术要求较高，限制了该策略在BRCA1传感分析中的应用。另一方面，单纯的内标策略无法满足实际复杂体系中BRCA1基因序列检测灵敏度的需求。基于此，本项目拟将内标策略、信号叠加策略和酶切循环放大技术等多种传感策略整合起来，发展新型超灵敏、叠加内标型BRCA1基因电化学传感器。所建立的叠加内标传感策略不仅有望显著改善电化学传感器的重复性，还可大幅度降低BRCA1基因序列的检测限。

对乳腺癌易感基因(BRCA1)的检测分析在促进乳腺癌的早期临床诊断和治疗方面具有深远意义。已报道的BRCA1基因传感器，其检测数据的准确性和可靠性易受仪器光源状态、实验环境、实际样品中的复杂成分等外界因素影响，严重制约了BRCA1基因传感器的发展。针对以上问题，本项目开发了一种金属纳米团簇荧光探针，以此来构筑传感器用于BRCA1基因的灵敏检测。通过合理设计DNA模板链的序列及结构，合成了具有荧光发射的DNA介导铜纳米团簇；研究了DNA链的序列和它的二级结构对铜纳米团簇发射峰波长和强度的调控机制；结合微流控技术，降低了传感器的制备成本；研究了发射峰的荧光信号随BRCA1基因浓度的变化规律，并以此发展了基于信号减弱模式的荧光传感策略，实现了BRCA1基因的灵敏检测。本项目所取得的科研成果为乳腺癌的早发现、早诊断和早治疗提供了有效的分析技术和分析手段。