模块化设计多功能蛋白纳米线及超灵敏生物传感应用

There is substantial interest in the use of biosensing to detect pathogen at ultralow levels. Recently, nanomaterials have been widely used in biosensing and have substantially improved sensitivity. However, most of these materials require functional biomolecular modification before they can be applied in biosensors. A new modularity multipurpose protein nanowire is designed in this application which is built by engineered self-assembly amyloid protein. The protein nanowire is composed by self-assembly components and functional elements that can be bound to each other through fused adapter peptide. The functional module is formed by functional elements binding to the surface of protein nanowire chassis which consist of self-assembly components. By controlling the self-assembly process, the resultant protein nanowire possesses a specific recognition module and a signal amplification module, allowing a dramatic increase of the signal when recognition module bind to target. Protein nanowire for different tasks can be easily built by combinating of different features. The modular designed multipurpose protein nanowire could be a series of universally applicable high-sensitivity biosensing systems. This method effectively integrates the different functions through modularity self-assembly. The new protein nanowire have more functionality options for different purpose. This protocol should provides a new route for developping protein nanodevice.

对病原分子进行高灵敏度检测一直是生物传感的重要需求之一。近年来，纳米材料在生物传感中的广泛应用，显著提高了病原检测的灵敏度。但无机纳米材料通常功能单一，需要功能生物分子修饰才能用于生物传感。本申请拟利用淀粉样蛋白自组装的性质和蛋白质工程技术，模块化设计可便捷更换功能配体的多功能蛋白纳米线。新的纳米线由自组装元件和功能元件构成。功能元件通过接头装置直接展示在自组装元件形成的通用纳米线底盘模块上成为功能模块。通过体外操纵自组装，可将特异性识别模块和信号放大模块整合在纳米线底盘上，得到同时具有特异结合能力和信号放大能力的多功能蛋白纳米线。将不同功能模块组合，多功能纳米线可适用于多种形式和不同目标分子的生物传感，成为普遍适用的系列化高灵敏蛋白纳米线传感器件。该方法利用模块化设计和分子自组装有效整合多种功能配体，使纳米线具有更多的功能选择以完成不同的任务，也为发展多功能纳米器件提供了新的技术路线。

发展高灵敏、高通量、快速的病原分析方法，在疾病早期诊断、有效防控和及时处置中具有重要意义。本研究针对当前分析技术中灵敏度不足等问题，利用蛋白的自组装现象，建立了体外自组装操控和自组装体多功能化的新策略，构建了几种多功能蛋白纳米器件：自组装荧光纳米线、蛋白纳米线3D探针和尺寸可调自组装酶纳米复合物。并将其用于不同类型的免疫分析，大幅提高了免疫分析的灵敏度。.1）自组装荧光纳米线，将大量荧光信号整合与少量结合分子，用于多目标病原筛查芯片，灵敏度相较常规方法提高了100倍以上；.2）蛋白纳米线3D探针，利用纳米线的空间立体构象，制备了分子取向可控的分子高密度固定三维结构，其所形成的多价效应提升了对目标分子的捕获能力，将病原检测灵敏度提高了1000倍，并将整个免疫检测过程控制在15分钟之内；.3）针对自组装体尺寸控制难题，建立了尺寸可控的自组装技术，构建了尺寸可调的酶纳米复合物，在不改变现有免疫分析步骤和仪器的情况下，大幅提高了心肌肌钙蛋白检测灵敏度达10000倍以上。