Nanopore-based multi-target analysis of Zika virus infection

主要研究者/项目负责人：施密特，霍尔格 项目摘要 受个性化医疗爆炸式增长的刺激，个性化医疗需要高度敏感的 基因组和蛋白质组学分析，单分子检测正在迅速发展， 基础研究技术成为先进分子诊断的基础 技术.尽管光学荧光检测曾经是首选的方法， 最近，当单个分子被拉过纳米孔时， 获得了极大的关注，特别是在下一代测序的背景下。直接的， 使用纳米孔的宽范围分子的无标记检测具有更广泛的潜力。 然而，充分利用纳米孔检测的灵敏度的主要障碍是 纳米孔的微小静电捕获体积和纳米孔的低静电捕获体积之间的不匹配是可能的。 在实际应用中遇到的浓度，例如在分子诊断中。 该项目的目标是克服这一限制与一种新颖的，创新的方法，以高- 使用单分子纳米孔分析的通量分子诊断。具体 本申请的目的是通过以下方式增加纳米孔的捕获速率（通量 数量级，并使用寨卡病毒（ZIKV）的临床样本验证这种方法 感染我们的中心假设是，这可以通过递送靶分子来实现 在孔的捕获半径内的微珠上使用基于芯片的光阱分离， 基于波导的光流控芯片。 本申请的目的将通过以下具体目标实现：（1） 使用载体珠的光学捕获提高纳米孔捕获率。这一目标将 验证了我们新方法的核心，并证明了两种核酸的50，000倍改进 酸和蛋白质;（2）芯片上集成的微流体样品处理与高 在超低（阿托摩尔）浓度下的通量分子检测;和（3）多重直接 从复杂的样品基质（血清、唾液、尿液、精液）开始检测ZIKV感染。 所提出的工作的创新性贡献是：（i）载体颗粒的光学捕获， 分子靶向递送;（ii）为基于纳米孔的， 不同分子生物标志物的无标记检测;和（iii）纳米孔的临床验证- 基于多种分析物类型的分析。拟议的工作意义重大，因为它将 介绍第一个集成系统，匹配的灵敏度，简单性和多功能性， 单分子纳米孔检测与分子诊断的现实世界的限制。作为 因此，这种方法将适用于许多分子靶标和应用。 1

Principal Investigator/Program Director: Schmidt, Holger Project Summary Spurred by the explosive growth in personalized medicine which requires highly sensitive genomic and proteomic analysis, single molecule detection is rapidly developing from a fundamental research technique into the foundation for advanced molecular diagnostic techniques. Whereas optical fluorescence detection used to be the approach of choice, electrical detection of single molecules as they are drawn through a nanopore has recently gained great attention, particularly in the context of next generation sequencing. The direct, label-free detection of a wide range of molecules using nanopores has much broader potential. However, a major obstacle for fully exploiting the exquisite sensitivity of nanopore detection is the mismatch between the minute electrostatic capture volume of a nanopore and the low concentrations encountered in real-world applications, e.g. in molecular diagnostics. The goal of this project is to overcome this limitation with a novel, innovative approach to high- throughput molecular diagnostics using single molecule nanopore analysis. The specific objectives of this application are to increase the capture rate (throughput) of a nanopore by orders of magnitude and to validate this approach with clinical samples for Zika virus (ZIKV) infection. Our central hypothesis is that this can be accomplished by delivering target molecules isolated on microbeads within the capture radius of the pore using a chip-based optical trap on a waveguide-based optofluidic chip. The objectives of this application will be accomplished by the following Specific Aims: (1) Nanopore capture rate enhancement using optical trapping of carrier beads. This Aim will validate the core of our new approach and demonstrate a 50,000x improvement for both nucleic acids and proteins; (2) On-chip integration of microfluidic sample processing with high throughput molecular detection at ultralow (attomolar) concentrations; and (3) Multiplex direct detection of ZIKV infection starting from complex sample matrices (serum, saliva, urine, semen). The innovative contributions of the proposed work are: (i) Optical trapping of carrier particles for molecular target delivery; (ii) Creating an integrated chip-based system for nanopore-based, label-free detection of diverse molecular biomarkers; and (iii) Clinical validation of nanopore- based analysis of multiple analyte types. The proposed work is significant because it will introduce the first integrated system that matches the sensitivity, simplicity, and versatility of single-molecule nanopore detection with the real-world constraints for molecular diagnostics. As such, this approach will be applicable to many molecular targets and applications. 1