基于酶催化和化学选择性链接的食品脂多糖分析方法研究

As an endotoxin produced by the lysis of Gram-negative bacteria, lipopolysaccharides (LPS) is a safe trouble in human daily diet. The content of LPS in the food product can reflect hygiene quality of the materials and the production process. However, the current methods for detecting LPS have the serious limitation. This aim of this project is to develop an innovatively strategy to overcome the shortages that a variety of LPSs cannot be simultaneously recognized using the current methods. The strategy displayed in this project will be multi-targeted and can realize the sensitive analysis of LPS. According to chemical properties of LPS, this project will develop a novel combined method of enzyme catalysis and chemoselective ligation to capture and detect three constituent parts (lipid A, core polysaccharide, and O-specific sidechain) of LPS. Specifically, both core polysaccharide and O-specific sidechain will be covalently linked through oxmie or hydrozone separately derived from the hydroxylamine or hydrazine reaction with the oxidized products which can be obtained from LPS oxidation catalyzed by galactose oxidase and its variants. At the same time, lipid A can be bound effectively with antibacterial polypeptides, thereby realizing the multi-targeting recognization and the following chemical detection. Moreover, this project will enhance the sensitivity of LPS assay by use of signal amplification nanotechnology. Implementation of this project will make up the studying blank related to the enzyme catalysis and chemoselective ligation of LPS and other relevant research issues, solve the scientific event about simultaneous multi-targeting capture of different LPSs, and provide the important research basis for the establishment of new strategy for the detection of total LPS in the food samples.

脂多糖（LPS）作为革兰氏阴性菌裂解产生的内毒素，是日常饮食的安全隐忧。食品成品中LPS的含量高低能反映食品原料和加工过程的卫生质量，但其检测方法仍具有较大局限性。本项目拟克服现有手段无法同时识别多种LPS的缺陷，创新性地提出多靶向检测新策略，实现食品总LPS的灵敏分析。将根据LPS的化学特征，发展酶催化和化学选择性链接相结合的方法，探索其三个组成部分（类脂A、核心糖和O-特异侧链）的捕获。将利用半乳糖氧化酶及变异体催化LPS氧化，并利用其氧化产物与羟胺或肼反应产生的肟或腙共价结合核心糖和O-特异侧链两个部分。另外，利用抗菌肽高效结合类脂A部分，实现对LPS的多靶向识别。将进一步采用纳米技术放大信号，提高LPS检测的灵敏度。本项目的实施将弥补LPS的酶催化和化学选择性链接研究的空白，有望解决多种LPS的同时多靶向捕获的科学问题，为食品中总LPS分析新策略的建立提供重要研究基础。

脂多糖（LPS）作为革兰氏阴性菌裂解产生的内毒素，是日常饮食的安全隐忧。食品成品中LPS的含量高低能反映食品原料和加工过程的卫生质量，但其检测方法仍具有较大局限性。本项目克服现有手段无法同时识别多种LPS的缺陷，创新性地构建了pH敏感的脂质双层介导离子运输的比色检测法、基于三维金纳米颗粒/二茂铁/脂质体纳米簇的电化学检测法、腙化学辅助DNAzyme的荧光分析方法、腙化学辅助三维DNA行走机器的荧光分析方法、基于CRISPR/Cas体系的荧光分析方法和基于CRISPR/Cas体系偶联DNA行走机器的荧光分析方法，实现食品总LPS的简单、快速、准确和灵敏分析。在项目研究过程中，已正式发表期刊论文 27 篇（其中 25 篇被 SCI收录），申报发明专利 5 项（授权 1 项），招收硕士研究生12人（毕业7人），培养博士研究生3人（毕业1人）。另外，还出版了英文专业书籍的4个章节，获得了的5项奖励。本项目的实施弥补LPS的酶催化和化学选择性链接研究的空白，解决多种LPS的同时多靶向捕获的科学问题，为食品中总LPS分析新策略的建立提供了重要研究基础。