Detection of Category A Pathogens by Gold Nanoparticles

金纳米粒子检测 A 类病原体

• 批准号: 7118138
• 负责人: Steven M Wolinsky
• 金额: $ 113.13万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7118138
• 关键词: Bacillus anthracis; Raman spectrometry; bacterial DNA; bacterial RNA; biohazard detection; biomedical equipment development; biotechnology; bioterrorism /chemical warfare; cooperative study; gold; microarray technology; molecular probes; technology /technique development

DESCRIPTION (provided by applicant): Recent deliberate exposure of civilian population of the United States populace to Bacillus anthracis spores uncovered an unmet need for tests to rapidly diagnose disease caused by this bacterium. A major challenge for rapid and accurate pathogen detection is the development of methods that do not rely on the polymerase chain reaction (PCR) or comparable target- or signal-amplification systems to minimize instrumentation and reagent needs and enable point-of-care or field testing. We propose to develop a highly selective sequence-specific, chip-based method for detection of DNA from B. anthracis by exploiting the unique properties of DNA-gold nanoparticle conjugates. The proposed methodology promises to be far simpler than current methods and obviates the need for large-scale analytical instrumentation and trained personnel. Preliminary data show that detection assays based on these novel nanostructures are substantially more sensitive and selective than conventional assays based on fluorophore-labeled DNA probes. Ultimately, the assay will be as accurate and sensitive as PCR-based assays. By combining gold nanoparticles with Raman spectroscopic fingerprints, we can effectively distinguish dissimilar polynucleotide sequences of multiple infectious pathogens in the same sample without target sequence amplification. In contrast to fluorescence-based hybridization, gold nanoparticle probes labeled with oligonucleotide and Raman-active dyes offer several advantages, including higher potential for multiplexing and orders-of-magnitude higher sensitivity and selectivity. We propose to implement this assay in a simple microfluidic device with integrated capacity for specimen processing, DNA/RNA purification, separation, hybridization, and signal detection, thus offering the possibility for integrated, high-throughput screening in a "lab-on-a-chip," with easily-read outputs. The overall goal of this proposal is to create a rapid, sensitive, specific, and cost-effective tool for detection and identification of the category A pathogen, B. anthracis, and a battery of other pathogens with similar clinical syndromic presentation using a single sample. In Specific Aim 1, we will develop a rapid, high-sensitivity, high-selectivity sequence-specific test based on the optical, hybridization, and catalytic properties of gold nanoparticle DNA conjugates and Raman-active dyes for the simultaneous detection of multiple target polynucleotide molecules. In Specific Aim 2, we will design, develop, and fabricate a flexible polynucleotide array-based microfluidic platform with cell and analyte handling capabilities for cost-effective, high-throughput, selective detection of captured target DNA and RNA bound to nanoparticles capable of surface-enhanced Raman scattering (SERS). Lastly, in Specific Aim 3, we will establish proof-of-principle and validate the performance of the array-based multiplex detection of conserved polynucleotides derived from anthrax and other infectious pathogens with a similar respiratory syndromic presentation at the point-of-care.

描述（由申请人提供）：最近美国平民故意接触炭疽芽孢杆菌孢子，发现对快速诊断由这种细菌引起的疾病的测试的需求未得到满足。快速准确的病原体检测的一个主要挑战是开发不依赖聚合酶链式反应 (PCR) 或类似的靶标或信号放大系统的方法，以最大程度地减少仪器和试剂需求并实现护理点或现场测试。我们建议开发一种高度选择性的序列特异性、基于芯片的方法，通过利用 DNA-金纳米粒子缀合物的独特性质来检测炭疽芽孢杆菌的 DNA。所提出的方法有望比现有方法简单得多，并且不需要大型分析仪器和训练有素的人员。初步数据表明，基于这些新型纳米结构的检测方法比基于荧光团标记 DNA 探针的传统检测方法具有更高的灵敏度和选择性。最终，该检测将与基于 PCR 的检测一样准确和灵敏。通过将金纳米颗粒与拉曼光谱指纹相结合，我们可以有效地区分同一样本中多种传染性病原体的不同多核苷酸序列，而无需目标序列扩增。与基于荧光的杂交相比，用寡核苷酸和拉曼活性染料标记的金纳米颗粒探针具有多种优势，包括更高的多重检测潜力以及数个数量级的更高灵敏度和选择性。我们建议在一个简单的微流体装置中实施这种测定，该装置具有样本处理、DNA/RNA纯化、分离、杂交和信号检测的集成能力，从而为在“实验室上”进行集成、高通量筛选提供了可能性。 -chip”，具有易于读取的输出。该提案的总体目标是创建一种快速、灵敏、特异且经济有效的工具，用于使用单个样本检测和鉴定 A 类病原体、炭疽芽孢杆菌以及具有类似临床症状表现的一系列其他病原体。在具体目标1中，我们将基于金纳米粒子DNA缀合物和拉曼活性染料的光学、杂交和催化特性开发一种快速、高灵敏度、高选择性的序列特异性测试，用于同时检测多个目标多核苷酸分子。在具体目标 2 中，我们将设计、开发和制造基于多核苷酸阵列的灵活微流控平台，该平台具有细胞和分析物处理能力，可对捕获的目标 DNA 和 RNA 进行选择性检测，这些目标 DNA 和 RNA 与能够表面吸附的纳米粒子结合。 -增强拉曼散射（SERS）。最后，在具体目标 3 中，我们将建立原理验证并验证基于阵列的多重检测的性能，该检测对来自炭疽和其他具有类似呼吸综合征表现的传染性病原体的保守多核苷酸进行护理。