基于核酸适体结构开关和酶切循环信号放大的抗生素多残留分析研究

Aptamer is a kind of molecular recognition element which can combine with its target with high affinity and specificity. The key technology in aptamer-based biosensors lies in how to translate the recognition mechanism into a detectable signal. In this project, structure-switching aptamers would be designed aiming at the analysis of common antibiotic residues in animal derived food, which utilizes the specific recognition of aptamers and the advantages of fluorescence resonance energy transfer (FRET) between quantum dots and metal nanoparticles. Magnetic nanoparticles/quantum dots and metal nanoparticles are respectively used to modify the complementary DNA fragments. The specific binding of the target to the aptamer could open the aptamer-complementary DNA double strands to alter the FRET efficiency. Thus, in combination with the separation and enrichment of magnetic nanoparticles and Exo III enzymatic circulation signal amplification technology, the “turn-on” fluorescence detection of antibiotic residues would be established. The multiple FRET will be explored to bulid using quantum dots - metal nanoparticles with significantly different optical properties, in order to realize simultaneous detection of many kinds of antibiotic residues. Moreover, researches will be focused on the structure transformation of structure-switching aptamers in the process of target recognition, and the different mechanism and conformational changes involved in the specific recognition of aptamers with the antibiotic targets. This project would be helpful to clarify the design and working mechanism of structure-switching aptamers for small molecules. It has important significance for the the versatility extension of the design strategy of structure-switching aptamers, and will contribute to the application of aptasensors in the detection of hazard factors in complex food.

核酸适体是能对其靶标进行高亲和力和强特异性结合的分子识别元件，其传感技术的关键是如何将适体的识别机理转变为可检测信号。本项目针对动物性食品中常见抗生素残留，结合适体特异性识别和量子点-金属纳米粒子荧光共振能量转移（FRET）的优势，设计核酸适体结构开关。用磁性纳米粒子/量子点和金属纳米粒子分别修饰互补核酸片段，适体与靶标的特异性结合打开适体-互补核酸双链从而改变FRET效率，结合磁性纳米粒子的分离富集和Exo III酶切循环信号放大，建立“turn-on”荧光法检测抗生素；探索利用光谱性质显著差异的量子点-金属纳米粒子构建多重FRET，实现抗生素多残留分析；研究目标物识别过程中适体开关的结构转换，探索核酸适体特异性识别抗生素靶分子的作用机理及构象变化。本研究阐明小分子核酸适体结构开关的设计和工作机制，对拓展设计策略的通用性有重要意义，为将适体传感器用于检测复杂食品基质中的危害因子奠定基础。

核酸适配体是能对其靶标进行高亲和力和强特异性结合的分子识别元件，利用核酸适配体结构开关性质可以构建传导靶分子识别的通用技术。本项目通过创新核酸适配体结构开关的设计及分子识别的信号转换放大策略，建立了一系列简便、快速、高灵敏的荧光传感器，用于检测抗生素等小分子和重金属。主要工作如下：（1）基于荧光共振能量转移原理，设计具有通用性的双螺旋适配体结构开关，成功检测牛奶中的卡那霉素和氯霉素。基于适配体的特异性识别，构建特异性强、稳定性高的三螺旋DNA分子开关检测啶虫脒，在不需要改变三螺旋分子结构的前提下，通过改变适配体推广用于检测其他目标物。（2）基于适配体结构转换，结合杂交链式等温扩增和双荧光染料协同作用进行信号放大，利用磁性分离技术和石墨烯猝灭作用降低背景信号，构建检测牛奶中妥布霉素的高灵敏非标记荧光传感器。（3）利用适配体对目标物的高亲和性，及适配体结合目标物前后的结构转换，构建基于酶切循环信号放大的高灵敏度、高特异性荧光分析方法，用于检测土霉素和有害重金属铅离子，方法抗干扰能力强、检测效率高，设计巧妙且具有推广性。（4）利用目标物竞争性结合适配体使互补链释放的特点，基于T-Hg2+-T错配识别，设计了两种目标物打开结构开关引起荧光信号变化的传感体系检测Hg2+，分别利用磁性分离技术和稀土敏化发光，显著提高了方法的灵敏度，成功用于检测水体系和鱼中的汞离子，设计具有通用性和可推广性。（5）此外，以靶分子高特异性识别诱导适配体构象改变，以核酸染料为信号报告分子，建立非标记荧光传感器检测牛奶中的四环素。项目的研究结果丰富了核酸适配体结构开关光学传感器的设计策略，所建立的检测方法具有操作简单、耗时短、成本低、无需昂贵仪器等优势，实现了复杂食品基质中小分子和重金属的高灵敏、高特异性的定量分析。项目执行期间共发表SCI论文11篇，影响因子4.0以上5篇，另有4篇SCI论文在投，申请专利7项，获吉林省自然科学奖二等奖1项。