Nanoporous Gold: Extractive Substrate for High-Speed Ultrasensitive Bioassays

描述(申请人提供)：2002年，病毒夺走了近1500万人死于传染病的生命中的一个重要组成部分。病毒传播可以通过几种途径发生，包括接触受感染的人，摄入受污染的食物和水，或接触蚊子和扁虱等媒介。这些途径可能只需要传播几十到数百种病毒就能引发感染，随着我们这个星球的全球化和城市化进程的加快，这些途径只会变得更加普遍。因此，快速和非常低水平地检测这些和许多其他病原体和疾病标志物的能力对于公共健康监测、食品/水安全和生物恐怖主义来说是一个极具挑战性的命题。虽然最近的突破使得在固相捕获后能够以个位数和两位数的水平检测病毒和其他纳米目标(例如蛋白质)，但样品/标签孵化所需的时间仍然是过渡到监视/监测领域的瓶颈。这一建议旨在通过探索纳米多孔金(NPG)作为流动捕获底物的潜力来重新定义检测速度，以有效地提取病毒和其他同等大小的病原体和疾病标记物(例如抗体)，同时考虑到有效性能所需的其他考虑因素。这一策略的基础在于预测的传质速率的改善，因此结合速率，在多相分析中的捕获和标记步骤中，可以通过流过纳米孔材料来实现。模型预测，与已知最有效的方法相比，约束率可能会增加两个数量级以上。因此，计划进行两组实验，使用基于纳米金颗粒的表面增强拉曼散射(SERS)测量来评估这种可能性。在第一组实验中，不同孔径的NPG膜将被制备、衍生化，并作为模型病毒猫杯状病毒FCV的萃取相进行测试。FCV直径约30 nm，是一种有效的诺沃克病毒模拟物，将使对提取物的孔大小进行深入、系统的评估。这些研究还将测试流速对捕获和标签效率的影响，并共同为其他潜在应用中的性能优化提供一套预测规则。此外，还将进行实验，以最大限度地减少通过使用封闭剂进行的非特异性吸附的影响，以及通过结合样品预过滤器来减少膜堵塞的潜在并发症。在第二组实验中，这些指南将应用于检测几种基质中的FCV，包括全山羊血清、自来水和地下水。公共卫生相关性：这项赠款提案旨在通过探索纳米多孔金(NPG)膜作为捕获底物流动的潜力来重新定义异质免疫分析的速度，以快速、高效和选择性地浓缩纳米尺寸的病原体(例如病毒和蛋白质)。这一策略的基础在于：(1)通过流过纳米孔材料可以实现捕获和标记步骤的传质速率和结合率的预期改善；以及(2)使用修饰的金纳米颗粒和表面增强拉曼散射(SERS)的读出技术的高灵敏度。为了执行上述任务，我们召集了一支由犹他大学化学、化学工程和生物工程系以及亚利桑那州立大学材料学院的科学家和工程师组成的团队。

DESCRIPTION (provided by applicant): Viruses claimed a significant component of the nearly 15 million lives lost to infectious diseases in 2002. Viral transmission can occur through several routes, including contact with infected individuals, ingestion of contaminated food and water, or contact with a vector like mosquitoes and ticks. These pathways, which may only need to transmit a few tens to hundreds of viruses to trigger an infection, will only become more prevalent as the globalization and urbanization of our planet accelerates. Thus, the ability to detect these and many other pathogens and disease markers rapidly and at very low levels stands as an extremely challenging proposition central to pubic health monitoring, food/water safety, and bioterrorism. While recent breakthroughs have led to the capability to detect viruses and other nanometric targets (e.g., proteins) at single and double digit levels after capture on a solid phase, the time required for sample/label incubation remains a bottleneck for transitioning to the surveillance/monitoring arena. This proposal seeks to redefine assay speed by exploring the potential of nanoporous gold (NPG) to function as a flow-through capture substrate for the efficient extraction of viruses and other comparably-sized pathogens and disease markers (e.g., antibodies), while at the same time accounting for other considerations needed for effective performance. The basis for this strategy rests with predicted improvements in the mass transfer rates, and thus the binding rates, for both the capture and labeling steps in heterogeneous assays that may be realized by flow through a nanoporous material. Models project potential increases in binding rates of more than two orders of magnitude with respect to the most effective of the known approaches. Two groups of experiments are therefore planned to assess this possibility using gold nanoparticle-based surface enhanced Raman scattering (SERS) measurements. In the first group of experiments, NPG membranes of varied pore size will be fabricated, derivatized, and tested as flow through extraction phases for the model virus feline calicivirus, FCV. FCV, which has ~30-nm diameter and is an effective norovirus simulant, will enable an in-depth, systematic assessment of extraction with respect to pore size. These studies will also test the effect of flow rate on capture and label efficiency, and collectively will provide a set of predictive rules for performance optimization in other potential applications. In addition, experiments will be conducted to minimize the impact of nonspecific adsorption by use of blocking agents, as well as potential complications from membrane clogging through the incorporation of sample prefilters. In the second group of experiments, these guidelines will be applied to assays for the detection of FCV in several matrices, including whole goat serum, tap water and groundwater. PUBLIC HEALTH RELEVANCE: This grant proposal seeks to redefine the speed of heterogeneous immunoassays by exploring the potential of nanoporous gold (NPG) membranes to function as flow through capture substrates for the rapid, efficient and selective concentration of nanometrically-sized pathogens (e.g., viruses and proteins). The basis for this strategy rests with: (1) the predicted improvements in the mass transfer rates, and thus the binding rates, for both the capture and labeling steps that may be realized by flow through a nanoporous material; and (2) the high sensitivity of a readout technique that uses modified gold nanoparticles and surface enhanced Raman scattering (SERS). To carry out the above tasks, we have assembled a team of scientists and engineers from the University of Utah Departments of Chemistry, Chemical Engineering, and Bioengineering, and from the Arizona State University School of Materials.