Dye-loaded Nanoparticle Platform for Rapid and Sensitive Vivarium Pathogen Detection

项目摘要 这项建议开发了啮齿动物健康的超灵敏病原体检测系统的关键方面。 并响应NIH FOA PAR-21-225开发用于动物的新工具和装置 研究设施，并支持动物模型的护理。该项目的目标是开发一种微流体 直接从动物或被污染的床上快速、廉价地检测病原体的设备。该设备 将在笼侧使用，并将补充目前的啮齿动物健康监测计划，这些计划旨在 每季度对房间或笼架进行监控。该设备的使用将鼓励快速缓解和解决疫情 以及确认动物进口的健康状况。该平台的优势将是专用性， 简单性、速度和敏感度在“3R”一致的方法中。 检测系统的特异性依赖于设计的合成寡核苷酸的DNA杂交 特别针对感兴趣的病原体，其DNA来自直接从啮齿动物或从啮齿动物的感染源捕获的DNA 床上用品样品。检测步骤将是快速的，因为DNA杂交将不依赖于循环扩增 而是将灵敏度要求转移到从介孔二氧化硅中释放染料 纳米颗粒使用pH依赖的染料释放。在这份第一阶段提案中，平台的关键方面将是 对4个目标进行了可行性优化和测试。首先，将建立病原体DNA制备方案 使用细菌和病毒的DNA来源，以及对DNA收集的病原体检测限度进行量化。第二, 用于病原体检测的合成寡核苷酸将被优化并与磁性结合 纳米粒子。第三，介孔二氧化硅纳米颗粒(MSNPs)的内孔和外表面将 差示标记，使内表面充满可逆结合的染料分子，而 外表面覆盖有DNA，它将与病原体特异性检测寡核苷酸杂交。 最后，将病原体检测与超灵敏相结合，对平台的可行性进行了验证 对pH敏感的染料释放方面。该平台的创新之处在于利用DNA二级结构 提供病原体特异性，使用专为饱和染料负载而设计的具有pH敏感性的mSNPs 染料释放，以及使用DNA杂交将特异性和敏感性特征物理绑定在一起， 允许通过使用磁性纳米颗粒来丰富检测络合物。

Project Summary This proposal develops key aspects of an ultrasensitive pathogen detection system for rodent animal health surveillance and is in response to the NIH FOA PAR-21-225 to develop novel tools and devices for animal research facilities and to support the care of animal models. The goal of the project is to develop a microfluidic device for rapid and inexpensive pathogen detection directly from an animal or from soiled bedding. The device will be used cage-side and will complement current rodent health monitoring programs which are designed to monitor rooms or cage racks quarterly. Use of the device will encourage rapid outbreak mitigation and resolution as well as confirm health status for animal importation. The advantages of the platform will be specificity, simplicity, speed, and sensitivity in a “3Rs” consistent approach. The specificity of the detection system relies on DNA hybridization of a synthetic oligonucleotide designed specifically for the pathogen of interest with DNA captured from infectious agents directly from the rodent or from bedding samples. The detection step will be rapid since the DNA hybridization will not rely on cyclic amplification of the DNA but instead will shift the sensitivity requirements to release of dye from mesoporous silica nanoparticles using pH-dependent dye release. In this Phase I proposal, the key aspects of the platform will be optimized and tested for feasibility in 4 Aims. First, the pathogen DNA preparation protocol will be established using a bacterial and viral DNA source, and the limit of pathogen detection quantified for DNA collection. Second, the synthetic oligonucleotide that serves in pathogen detection will be optimized and bound to magnetic nanoparticles. Third, internal pores and external surfaces of mesoporous silica nanoparticles (mSNPs) will be differentially labelled so that the internal surfaces are saturated with reversibly bound dye molecules while the external surfaces are coated with DNA that will hybridize with the pathogen specific detection oligonucleotide. Lastly, proof of feasibility of the platform will be tested by combining the pathogen detection and ultrasensitive pH-responsive dye release aspects. The innovation of this platform is in the use of DNA secondary structure to provide pathogen specificity, the use of mSNPs designed specifically for saturated dye loading with pH-sensitive dye release, and the use of DNA hybridization to physically bind the specificity and sensitivity features together, allowing enrichment of the detection complexes with the use of magnetic nanoparticles.