基于表面增强拉曼散射活性纳米通道的DNA损伤单分子检测新技术研究

Studies on genetic toxicity of environmental pollutants urgently need a new generation of DNA damage detection technology. Nanopore is considered to be one of the most promising methods for the single-molecule DNA detection, whereas it cannot provide the DNA damage-related chemical structure information. Accordingly, in this project, a new single-molecule detection approach for DNA damages will be developed by preparing a novel kind of surface-enhanced Raman scattering (SERS)-active nanopores with comprehensive advantages of nanopore technology in single-molecule sensitivity and SERS technology in abundant molecule fingerprint. The project will be conducted as follows: firstly, solid-state nanopores suitable for the translocation of single DNA molecules will be prepared through focus ion beam, electrochemical etching, etc.; secondly, SERS activity will be combined into nanopores based on novel strategies of electromagnetic enhancement like optical nanoantennas integrated with concentric ring grating by using magnetron sputtering, surface modification, directed assembly, etc.; after that, a highly sensitive detection equipment will be set up and the responding signal analysis system will be developed. Then, different kinds of DNA damages, will be detected with the manufactured SERS-active nanopores to obtain their analysis models; at last, a novel nanopore-based single-molecule detection technology for DNA damages will be established. Therefore, the outcomes of this project will provide a new train of thought about the preparation of novel nanopores and a novel detection technology for DNA damages, thus providing new technique support for the related studies such as environmental toxicology, toxicity screening of chemicals, ect.

环境污染物遗传毒性等研究迫切需要新一代的DNA损伤检测技术。纳米通道是最具前景的DNA单分子检测技术之一，但目前的纳米通道技术难以直接给出DNA损伤相关的化学结构信息。本项目拟通过制备具有表面增强拉曼散射（SERS）活性的新型固体纳米通道，综合利用纳米通道适合单分子检测和SERS可提供分子结构指纹谱图的优点，研发DNA损伤单分子检测新技术。项目首先制备可调控单个DNA分子穿过的固体纳米通道，并利用纳米天线耦合同心环光栅、定向组装等策略使其具备高SERS活性且能同时产生离子电流和SERS响应；在此基础上，搭建高灵敏光/电检测装置、开发光/电信号综合分析系统，获取可给出损伤位点、损伤方式等信息的DNA损伤解析模型，从而建立基于SERS活性纳米通道的DNA损伤单分子检测新技术。项目研究成果可望为污染物致DNA损伤的无标记、高精准分析提供新的检测手段，进而为分子毒理分析等研究提供新的技术支持。

本项目围绕环境污染物遗传毒性等研究对新一代DNA损伤检测技术的迫切需求，开展了表面增强拉曼散射（SERS）活性石英玻璃纳米通道制备、DNA分子SERS检测体系探索、DNA高灵敏SERS检测方法研究等工作，建立了污染物致DNA损伤的无标记、高精准检测新技术。研究工作概述如下：.1. 利用石英玻璃优良的可塑性和抗变形能力，以石英毛细玻璃管为基体，采用激光拉制技术制备出孔径可控的石英玻璃纳米通道；在纳米通道表面通过定向组装等手段修饰金纳米颗粒，制备出SERS活性石英玻璃纳米通道。.2. 根据胍基化合物以及Zr4+与DNA磷酸骨架具有较强结合作用等机制，研究探索了DNA分子的SERS检测新体系，优化了相关检测条件，建立了DNA的高灵敏、高分辨SERS检测方法，进一步提升了SERS对DNA分子的检测性能。.3. 基于所建立的SERS检测体系与方法，构建了深度卷积神经网络分类模型，并将二者结合用于碱基差异的DNA样品分析，进而建立了基于SERS和机器学习的单碱基分辨DNA损伤检测新技术。 .4. 拓展研究思路，研究出系列生物酶的SERS检测新方法，发挥SERS光谱可以提供指纹谱图、灵敏度高以及抗光漂白等优点，实现了谷氨酰转肽酶、亮氨酸氨基肽酶等酶活性的高灵敏、高选择性检测。