基于DNA/PNA纳米技术的可调控传感界面构建及在ctDNA检测中的应用

As one of liquid biopsies, circulating tumor DNA (ctDNA) is tumor-derived fragmented DNA in the bloodstream released from cancer cells. The analysis of ctDNA provides a minimally invasive and specific way for early cancer diagnosis and prognosis. However, high sensitive and specific methods for detection of ctDNA are lacking. ctDNA analysis is still challenged in the application of liquid biopsies, since it is vital to differentiate circulating DNA from normal cells vs mutation-bearing sequences emerging from tumours. In this project, a new strategy based on DNA/PNA nanotechnology will be developed to detect ctDNA with high sensitivity and specificity. Due to DNA/PNA’s highly specific Watson-Crick base pairing, DNA and PNA molecules can be self-assembled into 3D tetrahedral nanostructures of various sizes with high predictability and precision. Therefore, a biosensing interface can be programmably and reproducibly engineered by using well-defined 3D DNA/PNA tetrahedral nanostructures within nanometer precision. By combining this controllable biosensing interface with label-free electrocatalytic assay based on Ru3+ adsorption and Fe3+ amplification, it is potential to develop a high sensitive and specific method for ctDNA detection. The neutral peptide nucleic acid (PNA) probe binds to DNA more strongly and specifically than DNA/DNA, due to the lack of electrostatic repulsion. Also, without charged phosphate groups, the amount of Ru3+ adsorption to PNA will be negligible, producing lower background current, thus improve signal-to-noise ratio. Moreover, the use of DNA clutch probes that prevent reassociation of denatured single-stranded DNA strands, and the use of clamp probes that eliminate cross-reactivity with wild-type DNA and with deliberately selected mutants, are able to sequester the high background of unmutated sequences and other closely sequences in the serum, and allow only the single ctDNA of interest to bind to DNA/PNA nanostructure probes with fast and specific response. By simply varing the size of the DNA/PNA nanostructures, the detection performances for ctDNA could be finely tuned. Therefore, this system offers the advantages of high sensitivity and specificity for ctDNA detection by an electrochemical readout. The successful implementation of the project will be helpful to facilitate the development of early cancer detection and evaluation of treatment effect.

循环肿瘤DNA（ctDNA）是由肿瘤细胞释放到外周血中的游离DNA，是液体活检对象之一。ctDNA检测具有低侵入性、特异性等优势，在癌症早期诊断及预后判断方面具有重要意义。如何区分游离DNA是来自正常细胞还是肿瘤细胞，实现高灵敏度、高特异性检测是其应用于液体活检的主要挑战。本项目拟利用DNA/PNA纳米技术，获得尺寸精确可控的DNA/PNA四面体纳米结构，结合表面修饰方法及免标记电化学信号放大法，构建纳米尺度精确可控的电化学传感器，发展高灵敏度、高特异性的ctDNA检测方法。无电荷的PNA，有利于提高识别靶标效率和特异性；降低背景电流，提高信噪比。通过加入“爪”和“钳”探针，分别用于防止热变性单链DNA复性和消除与野生型DNA交叉反应，有效降低野生型DNA干扰，暴露出单链靶ctDNA。可调控传感界面识别并调控该靶分子的检测性能。项目的成功实施将有助于癌症早期诊断、治疗效果评估等发展。

循环肿瘤DNA（ctDNA）是一种来源于肿瘤细胞的释放、并可在血液中被检测的肿瘤标志物，在肿瘤无创诊断和实时检测方面具有潜在的临床应用价值。但由于ctDNA含量低、血液环境复杂、来源正常细胞的游离DNA干扰大等，对实现ctDNA检测是个巨大挑战。本项目利用DNA纳米技术，设计并合成了系列横向尺寸一致、纵向高度可调的四面体框架核酸纳米结构，结合表面修饰技术，构建了可精确调控的传感界面用于系统地研究不同尺寸四面体框架核酸检测ctDNA的性能，实现了 ctDNA快速、灵敏的检测。结果表明，基于四面体框架核酸的传感界面，相比于同样具有间距可控的polyA探针组装传感界面，对靶标ctDNA具有更高的检测灵敏度。将仿生分子磷脂酰胆碱引入框架核酸传感界面，有利于提高框架核酸在界面的稳定性，实现了在血清中长时间信号稳定，并能够对靶标ctDNA快速响应，具有应用于临床检测的潜力。在此基础上，利用DNA纳米结构的精确可控性及界面有序自组装方法，结合光响应分子和无机纳米材料等，我们发展并构建了高性能的新型传感界面用于癌症早期多种肿瘤标志物检测，拓展了基于DNA纳米结构的纳米传感界面在肿瘤标志物相关检测中的应用。本项目的研究为高效性能传感界面的构建提供了理论基础和实验模型，为癌症的早期诊断和预后判断研究提供了新平台。