Nanopore-based multi-target analysis of Zika virus infection

首席研究员/项目总监：Schmidt, Holger 项目概要 受到需要高度敏感的个性化医疗爆炸性增长的推动 基因组和蛋白质组分析、单分子检测正在从 基础研究技术为先进分子诊断奠定基础 技术。尽管光学荧光检测曾经是首选方法， 最近，单分子通过纳米孔时的电检测 引起了极大的关注，特别是在下一代测序的背景下。直接的， 使用纳米孔对多种分子进行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精湛灵敏度的一个主要障碍是 纳米孔的微小静电捕获体积与低静电捕获体积之间的不匹配 实际应用中遇到的浓度，例如在分子诊断方面。 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，以实现高 使用单分子纳米孔分析进行通量分子诊断。具体的 该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) 的临床样本验证该方法 感染。我们的中心假设是，这可以通过传递目标分子来实现 使用基于芯片的光阱在孔捕获半径内的微珠上进行分离 基于波导的光流控芯片。 本申请的目标将通过以下具体目标来实现：(1) 使用载体珠的光捕获增强纳米孔捕获率。这个目标将 验证我们新方法的核心，并证明两种核酸均提高了 50,000 倍 酸和蛋白质； (2) 芯片上集成微流控样品处理，具有高 超低（阿摩尔）浓度下的通量分子检测； (3) 多路复用直接 从复杂的样本基质（血清、唾液、尿液、精液）开始检测 ZIKV 感染。 拟议工作的创新贡献是：（i）载体颗粒的光捕获 分子靶标递送； (ii) 创建基于芯片的集成系统，用于基于纳米孔的、 多种分子生物标志物的无标记检测； (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 推出第一个与灵敏度、简单性和多功能性相匹配的集成系统 单分子纳米孔检测与分子诊断的现实限制。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