核酸外切酶III辅助信号放大SERS传感界面构建及其检测DNA甲基转移酶的研究

DNA methyltransferase (DNMT) abnormal expression is closely related to many diseases. Therefore, the effective detection of DNMT activity is crucial to clinical diagnostics and drug screening. In this project, we plan to construct new SERS sensing interfaces based on exonuclease III-assisted signal amplification for highly sensitive detection of DNMT. Here, we design and synthesize probe DNA (hairpin DNA) with specific recognition sequence for DNMT to assure the selectivity. In the presence of DNMT, the probe DNA was methylated. Thus, it could be recognized and cleaved by restriction endonuclease. And then, based on the property of exonuclease III, which could selectively hydrolyze double-stranded DNA with specific structure and release residual DNA, a novel multiple signal amplification strategy was designed. This signal amplification strategy consisted of three recycling reactions in one reaction system. Combining the high sensitive quantitative analysis ability of SERS technology and the efficient separation characteristic of magnetic nanomaterial, a new SERS assay for detection of DNMT was developed based on exonuclease III-assisted signal amplification. This project could effective analysis of low level DNMT with high sensitivity, high selectivity, rapidity and simplicity. This project will not only provides new design ideas for detection of DNMT, but also provides powerful technique supports for DNMT related clinical diagnostics and drug screening.

DNA甲基转移酶(DNMT)异常表达与多种疾病密切相关，有效检测DNMT对疾病诊断和药物筛选意义重大。本项目拟构建核酸外切酶III辅助信号放大的表面增强拉曼散射(SERS)传感界面用于DNMT的高灵敏检测。通过设计合成含有识别序列的发夹型DNA作为识别探针，实现对DNMT的特异性识别；利用DNMT调控识别探针发生甲基化反应，协同限制性内切酶识别、切割甲基化识别探针，借助核酸外切酶III选择性水解特定结构双链DNA并释放剩余DNA片段的能力，构建集同一反应体系内三次循环反应为一体的“三重”信号放大策略；融合SERS技术高灵敏定量分析的优势和磁性纳米材料的分离特性，发展基于核酸外切酶III辅助信号放大的SERS传感新方法，实现对低含量DNMT高灵敏度、高选择性、简便快速的识别与检测。该项目的实施，不仅为DNMT的检测提供了新的思路，而且为DNMT相关的疾病诊断及药物筛选提供有力的技术支撑。

生物标志物的异常表达与多种疾病密切相关，因此有效检测生物标志物对疾病诊断和药物筛选意义重大。本项目以含有特定识别序列的发夹型DNA作为识别探针实现对生物标志物的高选择性识别；借助核酸外切酶选择性水解特定结构DNA的能力协同链杂交反应实现体系的信号放大；利用核酸适配体功能化磁珠实现体系的快速分离和富集；利用贵金属纳米材料优异的表面增强拉曼散射(SERS)增强性能构建SERS探针实现SERS信号高灵敏传导。据此，发展了信号放大SERS传感新方法用于生物标志物的检测。项目筛选、设计、合成了特异性识别DNA甲基转移酶（DAM）的含有“5´-G-A-T-C-3´”碱基序列的发夹型DNA识别探针，考察了DAM与识别探针的相互作用；构建了核酸外切酶协同链杂交信号放大策略，考察了不同类型核酸外切酶的作用机制；合成了不同种类的贵金属纳米材料，考察了不同纳米材料的SERS增强效果，同时优化了不同类型的拉曼报告分子；制备了磁控SERS传感器，测试了功能化磁珠的分离富集特性；优化了识别探针和SERS探针的浓度以及缓冲溶液的种类，探讨了核酸外切酶辅助信号放大SERS检测新机制，掌握了纳米材料形状和尺寸等因素对SERS探针性能及检测灵敏度的影响，拓宽了SERS分析技术的应用空间，为今后设计功能纳米材料SERS光学探针提供理论和实验依据。. 采用银包金纳米星作为SERS基底，协同核酸外切酶III辅助信号放大磁控SERS传感新方法，实现了对DAM的高灵敏、高选择性分析，检测的线性范围为5-140U/mL，检出限达0.15U/mL。该策略可用于不同DNA序列和拉曼报告分子的构建，进而能够简便的实现对多种生物标志物的同时分析，为解决生物标志物的低浓度、高背景快速检测难题提供了新的思路。. 共发表SCI论文7篇，其中TOP期刊SCI论文4篇；申请发明专利2项；会议论文3篇；联合培养硕士毕业生1人；项目组成员参加国内学术会议1次。