Nanopore-based multi-target analysis of Zika virus infection

首席调查员/项目主任：霍尔格·施密特 项目摘要 受个性化医疗爆炸性增长的刺激，个性化医疗需要高度敏感 基因组和蛋白质组分析，单分子检测正在从 为高级分子诊断奠定基础的基础研究技术 技巧。尽管光学荧光检测曾经是可选择的方法， 单分子被拉过纳米孔时的电子检测最近已经 获得了极大的关注，特别是在下一代测序的背景下。直接的， 利用纳米孔对广泛的分子进行无标记检测具有更广阔的潜力。 然而，充分利用纳米孔检测的精致敏感性的一个主要障碍是 纳米孔的微小静电捕获量与低静电捕获量之间的不匹配 在实际应用中遇到的浓度，例如在分子诊断中。 该项目的目标是通过一种新颖、创新的方法来克服这一限制，以实现 采用单分子纳米孔分析进行分子诊断。具体的 这项应用的目标是通过以下方式提高纳米孔的捕获率(吞吐量 数量级，并用寨卡病毒(ZIKV)的临床样本验证这种方法 感染。我们的中心假设是，这可以通过传递靶分子来实现。 使用基于芯片的光学陷阱隔离在孔的捕获半径内的微珠上 波导型光流控芯片。 本申请的目标将通过以下具体目标来实现：(1) 利用载体微珠的光学捕获提高纳米孔捕获率。这一目标将 验证我们新方法的核心，并展示两种核技术的50,000倍改进 酸和蛋白质；(2)微流控样品处理的芯片上集成 超低(全分子)浓度的通过量分子检测；以及(3)多重直接检测 从复杂的样本基质(血清、唾液、尿液、精液)开始检测寨卡病毒感染。 拟议工作的创新贡献是：(I)载流子粒子的光学捕获 分子靶向传递；(2)创建一个基于芯片的集成系统，用于纳米孔， 不同分子生物标志物的无标记检测；以及(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 推出首个集成系统，其敏感度、简单性和多功能性与 单分子纳米孔检测与分子诊断的现实世界约束。AS 这样，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