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

用于快速、灵敏检测动物病原体的负载染料的纳米颗粒平台

• 批准号: 10602993
• 负责人: Barbara Jean Stone
• 金额: $ 23.61万
• 依托单位: PARATECHS CORP.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2024-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10602993
• 关键词: Affect; Animal Model; Animal Welfare; Animals; Bacterial DNA; Beds; Binding; Biological Assay; Biomedical Research; Clinical; Collaborations; Collection; Complement; Complex; DNA; DNA amplification; Detection; Development; Device Designs; Device or Instrument Development; Devices; Disease; Disease Outbreaks; Dyes; Fluorescent Dyes; Funding; Funding Opportunities; Goals; Health; Health Status; Infectious Agent; Label; Laboratories; Laboratory Animal Production and Facilities; Letters; Linker DNA; Magnetic nanoparticles; Methods; Microfluidic Microchips; Microfluidics; Monitor; Oligonucleotides; Outcome; Pathogen detection; Periodicity; Personal Satisfaction; Phase; Physiology; Preparation; Protocols documentation; Research; Resolution; Rodent; Rodent Model; Sampling; Sensitivity and Specificity; Sentinel; Side; Silicon Dioxide; Site; Soil; Source; Specificity; Speed; Structure; Surface; System; Technology; Testing; Therapeutic; United States National Institutes of Health; Validation; animal care; design; detection platform; feasibility testing; improved; innovation; interest; magnetic beads; manufacture; nanoparticle; novel; outbreak control; pathogen; programs; response; skills; surface coating; tool; viral DNA

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.

项目概要 该提案开发了用于啮齿类动物健康的超灵敏病原体检测系统的关键方面 监测并响应 NIH FOA PAR-21-225 开发用于动物的新型工具和设备 研究设施并支持动物模型的护理 该项目的目标是开发微流体。 直接从动物或污染的床上用品中快速且廉价地检测病原体的装置。 将在笼子旁边使用，并将补充当前的啮齿动物健康监测计划，该计划旨在 每季度监测一次房间或笼架，使用该设备将有助于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在于特异性， “3R”一致方法中的简单性、速度和灵敏度。 检测系统的特异性依赖于设计的合成寡核苷酸的 DNA 杂交 专门针对感兴趣的病原体，从直接从啮齿动物或从传染源捕获的 DNA 由于 DNA 杂交不依赖于循环扩增，因此检测步骤将很快。 DNA 的变化，而是将灵敏度要求转移到从介孔二氧化硅中释放染料 使用 pH 依赖性染料释放的纳米颗粒 在第一阶段提案中，该平台的关键方面将是 优化并测试 4 个目标的可行性。首先，将建立病原体 DNA 制备方案。 使用细菌和病毒 DNA 来源，以及 DNA 收集的病原体检测限度。 用于病原体检测的合成寡核苷酸将被优化并与磁性结合 第三，介孔二氧化硅纳米粒子（mSNP）的内部孔和外表面将被 差异标记，使得内表面被可逆结合的染料分子饱和，而 外表面涂有 DNA，可与病原体特异性检测寡核苷酸杂交。 最后，结合病原体检测和超灵敏检测，验证该平台的可行性。 pH响应性染料释放方面，该平台的创新在于利用DNA二级结构来实现。 提供病原体特异性，使用专为 pH 敏感的饱和染料负载而设计的 mSNP 染料释放，以及使用 DNA 杂交将特异性和敏感性特征物理结合在一起， 允许使用磁性纳米颗粒富集检测复合物。