三链肽核酸分子信标结合纳米孔传感器用于生物标志物的同时检测研究

Biomarkers are a kind of substances which can be quantitatively measured and evaluated as an indicator for various physiological and biochemical processes. Plenty of studies have shown that a specific type of cancer is usually accompanied with the abnormal expression of a set of biomarkers, thus simultaneous detection of these biomarkers with high sensitivity and selectivity is of great value to the early diagnosis of cancers. Currently, there are mainly three types of methods to detect biomarkers, including ELISA, Western blot and immuno-PCR. In this project, we propose to realize the simultaneous detection of a group of biomarkers corresponding to a specific disease with high sensitivity and selectivity by the virtue of the combination of triplex-PNA molecular beacon (tMB) and nanopore sensor. Upon the interaction between the target and its corresponding tMB, the positively charged PNA signal transducing probe (barcode probe) would be released from the tMB. The barcode probes could translocate through alpha-hemolysin nanopore under negative transmembrane potential and produce characteristic current signals which could be used for qualitative and quantitative analysis. The coexisting negatively charged molecules such as DNA and DNA-protein complex would not produce interfering signals under this condition. By taking the advantage of asymmetric salt gradient, the sensitivity of this method could be greatly improved which would be suitable for the detection of biomarkers in real-world samples. Through the proposed strategy, we aim to establish a highly sensitive and selective method for the simultaneous detection of biomarkers which could find applications in clinical tests.

生物标志物是一类可在众多生理、生化过程中监测并可用于指示相应过程发生的物质的总称。研究发现人体内某些生物标志物的表达水平突然异常通常会伴随着某种特定疾病的发生，因此对这些相关生物标志物的高灵敏度、同时检测对于肿瘤等恶性疾病的早期诊断意义重大。目前检测生物标志物的方法主要包括酶联免疫吸附法、蛋白质印迹法及免疫PCR法等。本项目提出将纳米孔单分子传感器与三链肽核酸分子信标联用从而实现对同一疾病相关的多种生物标志物的高灵敏度、同时检测。待测物与对应的信标识别作用后释放出的带正电肽核酸信号输出分子（编码分子）在负电压驱动下穿越溶血毒素纳米孔产生的特征信号可用于待测物的定性、定量分析，并且完全不受DNA等带负电共存分子的干扰。通过构建不对称盐浓度梯度将极大地提高方法的灵敏度，从而进一步降低检测限。本项目的成功实施将建立一种高灵敏度、高特异性的生物标志物同时检测方法，并且可将该方法用于临床样本检测。

生物标志物检测对于肿瘤等恶性疾病的早期诊断具有重要意义；纳米孔检测技术具有快速、灵敏、高通量等优势，且低成本微型化的纳米孔检测仪器目前已经商业化，这些进步将极大地推动纳米孔技术在疾病诊断与即时检测领域中的应用。由于纳米孔检测是一种随机检测方法，即检测池中所有的分子都有可能被纳米孔捕获，因此无关分子频繁占据纳米孔将严重影响检测方法的灵敏度、准确度以及重现性。本项目提出构建三链肽核酸分子信标用于各类生物标志物的高灵敏度、高选择性检测研究。三链肽核酸分子信标由底物识别分子（DNA分子）和信号输出分子（肽核酸分子）构成。由于DNA 具有高度的可修饰性及功能集成性，因此三链肽核酸分子信标可通过抗原-抗体免疫反应、核酸适体识别以及碱基互补配对等生物分子相互作用实现对各类生物标志物的识别与响应。底物分子与信标分子结合后导致信标分子解体进而释放出用于标志物定量检测的肽核酸信号输出分子。由于该信号输出分子通过特殊设计而带正电，因此将在负电压驱动下穿越纳米孔产生信号；而结合了待测物的DNA分子由于带负电无法穿越纳米孔，因此避免了无关分子频繁占据纳米孔的问题从而大大优化了检测性能。为了进一步提升检测体系性能，我们使用野生型MspA纳米孔来代替alpha溶血毒素纳米孔，这是由于该纳米孔内壁带有大量的负电荷而对带正电的肽核酸分子有极高的捕捉率同时强烈排斥带负电荷的DNA，因此即便不采用盐浓度梯度策略也可以实现标志物的飞摩尔级别检测。

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