Nanoporous Gold: Extractive Substrate for High-Speed Ultrasensitive Bioassays

描述（由申请人提供）：在2002年因传染病丧生的近1 500万人中，病毒夺去了很大一部分生命。病毒传播可通过几种途径发生，包括与受感染个体接触，摄入受污染的食物和水，或与蚊子和蜱虫等媒介接触。这些可能只需要传播几十到几百种病毒就能引发感染的途径，随着我们星球全球化和城市化的加速，它们只会变得更加普遍。因此，在非常低的水平上快速检测这些病原体和许多其他病原体和疾病标志物的能力，对于公共卫生监测、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。虽然最近的突破已经导致能够在固相捕获后以一位数和两位数水平检测病毒和其他纳米目标（例如蛋白质），但样品/标签培养所需的时间仍然是过渡到监视/监测领域的瓶颈。该提案旨在通过探索纳米孔金（NPG）作为流动捕获底物的潜力来重新定义分析速度，以有效提取病毒和其他同等大小的病原体和疾病标志物（例如抗体），同时考虑到有效性能所需的其他考虑因素。该策略的基础在于预测的质量传递率的改进，从而结合率，在非均相分析中捕获和标记步骤可能通过流动通过纳米多孔材料来实现。与最有效的已知方法相比，模型预估结合率可能增加两个数量级以上。因此，两组实验计划利用基于金纳米粒子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，将制作不同孔径的NPG膜，衍生化，并在模型病毒猫杯状病毒（FCV）的提取阶段进行流动测试。FCV直径约为30nm，是一种有效的诺如病毒模拟物，可以对萃取物的孔径进行深入、系统的评估。这些研究还将测试流速对捕获和标记效率的影响，并共同为其他潜在应用的性能优化提供一套预测规则。此外，将进行实验，以尽量减少使用阻断剂的非特异性吸附的影响，以及通过加入样品预过滤器引起的膜堵塞的潜在并发症。在第二组实验中，这些指南将应用于几种基质（包括全羊血清、自来水和地下水）中FCV检测的分析。公共卫生相关性：本拨款提案旨在通过探索纳米多孔金（NPG）膜的潜力来重新定义异质免疫测定的速度，该膜可作为通过捕获底物的流动，用于快速、高效和选择性地浓缩纳米级病原体（如病毒和蛋白质）。该策略的基础在于：(1)通过纳米多孔材料的流动可以实现捕获和标记步骤的传质速率和结合速率的预测改进；(2)使用修饰金纳米粒子和表面增强拉曼散射（SERS）的读出技术的高灵敏度。为了完成上述任务，我们召集了来自犹他大学化学系、化学工程系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.