表面增强拉曼散射及成像技术对铜绿微囊藻细胞内微囊藻毒素的识别与分布研究

According to the demand and development trend of MC-LR toxin detection in Microcystis cells, and the traditional detection method is not sensitive, long period and destructive. To solve the above problems, this study proposed construction of several novel SERS targeted probes for the detection of MC-LR in Microcystis aeruginosa by the aptamer combined with nanotechnology, and carry out the research on SERS and spectral imaging for MC-LR. The main contents include: 1) study the preparation of new SERS active substrates based on graphene / noble metal nano materials; 2) to explore the design mechanism and preparation for the new type of SERS nano probe, which is based on aptamer and the nano material; 3) study SERS and spectral imaging on different targeting SERS probe for MC-LR standard samples and MC-LR in algae cells. The implementation of this project can lay the foundation of SERS and imaging technology for the realization of high selectivity and high sensitive detection of MC-LR, and it also provide the basis for the research of MC-LR content distribution of algae cells under different growth environmental conditions, and exploring the environmental impact mechanism for MC-LR produced. To reveal the relationship between the SERS probe interface reaction and recognition performance, to realize the function of design and performance control, ultimately to provide scientific basis for MC-LR pollution early warning and water pollution control measures.

针对产毒微囊藻细胞内MC-LR检测需求和发展趋势，传统检测方法对其检测存在不灵敏、周期长且具有破坏性等不足。针对上述问题，本项目提出利用核酸适配体与纳米技术相结合，构建几种针对铜绿微囊藻内MC-LR检测的新型SERS靶向探针，开展MC-LR的SERS及光谱成像研究。研究内容包括：1）基于石墨烯/纳米贵金属材料的新型SERS活性基底制备；2）探索针对核酸适配体和纳米材料的新型SERS纳米探针的设计机理与制备；3）不同靶向SERS探针对MC-LR标样及藻细胞内MC-LR的SERS及光谱成像。本项目的实施可为SERS及成像技术实现对MC-LR的高选择性、高灵敏检测奠定基础，也为研究MC-LR在不同环境条件下生长藻细胞内含量分布，探索MC-LR产生的环境影响机制提供依据。揭示SERS探针界面反应和识别性能之间的关系，实现功能设计与性能调控，最终实现对水体MC-LR污染预警与控制措施等提供科学依据。

微囊藻毒素-LR（MC-LR）是已知众多毒素种类中出现频率最高、毒性最强的一种淡水蓝藻毒素，MC-LR对人和动物的饮用水安全以及水生态系统均已构成严重的威胁，研究微囊藻毒素的合成分泌机制，控制有毒水华是当今世界关注的热点问题。本项目针对微囊藻毒素的高灵敏检测，构建不同的SERS基底和检测方案，结合多元判别分析方法，实现微囊藻毒素低至10-6 mg/L的痕量检测；针对纳米粒子对适配体探针的酶切保护作用，开展了基于针尖纳米孔的荧光检测技术，在荧光显微镜下直接观察DNA酶对适配体的实时监测，并将其应用于微囊藻毒素的高灵敏检测；进一步的以三峡库区的常见水华藻为研究对象，基于二维相关拉曼光谱分析技术，研究不同环境因子条件（如光强、氮限制、水体扰动等）对微囊藻细胞生长的促进或抑制机制，分析微囊藻细胞的拉曼特征指纹峰胡萝卜素的演变规律，预测藻细胞生长状态，为淡水水华微囊藻及毒素爆发预警提供基础依据。