PicoSep - A Microfluidic Single Molecule Free Solution Hydrodynamic Separation Pl

描述（由申请人提供）：微流控单分子自由溶液流体动力学分离平台尽管现代方法如微阵列、数字PCR和深度测序具有强大的功能，但凝胶电泳仍然是分子生物学的主力。虽然可以使用电泳获得高尺寸分辨率，但动态范围有限，需要针对小DNA（PAGE）、中等DNA（琼脂糖平板）或大DNA（PFGE）定制的特定测定。此外，检测灵敏度有限，定量准确性差，需要大量的样品体积和DNA量。毛细管电泳的特点是增加了灵敏度，快速分析时间，减少了样品消耗，但增加了复杂性，成本和相同的有限的尺寸动态范围。单分子自由溶液流体动力学分离（SML-FSHS）是一种基于尺寸的DNA分离和通过单分子计数进行定量的集成技术，其仅用缓冲液填充的微毛细管和压力源进行分离。该方法具有宽动态范围（100 bp - 27 kbp）、高尺寸分辨率和接近零的样品消耗（10 pL）的独特组合。此外，它是最灵敏的，无扩增的检测方法之一，只需要10-23摩尔的DNA进行分析，并拥有接近分子散粒噪声所施加的限制的定量准确性。在这个第一阶段的计划中，将开发一个名为PicoSep的微流体SML-FSHS平台，它超越了以前的微毛细管系统和任何当前DNA大小测定方法的大小测定性能，检测灵敏度和速度。这将通过以下方式实现：1）微/纳流体设备的初步演示，以提高速度和分辨率，2）新的结合电动样品堆叠，以进一步提高浓度灵敏度，以及3）开发紧凑的仪器平台，以降低成本并便于使用。该仪器将解决单分子方法体积大、昂贵和困难的先入之见。PicoSep无与伦比的灵敏度、宽动态范围和低样品消耗将使其成为临床诊断和癌症医学中无PCR、多重生物标志物分析的理想检测平台。 公共卫生相关性：微流控单分子自由溶液流体力学分离平台DNA的大小和分离是分子生物学中最基本和最广泛使用的工具之一。PicoSep将具有比任何现有技术更宽的尺寸动态范围、更高的检测灵敏度和更低的样品消耗。这将使开发新的高灵敏度，多重生物标志物测定用于临床诊断和癌症研究，比目前的方法更强大，更便宜。

DESCRIPTION (provided by applicant): A Microfluidic Single Molecule Free Solution Hydrodynamic Separation Platform Despite the power of modern methods such as microarrays, digital PCR, and deep sequencing, gel electrophoresis remains the workhorse of molecular biology. While high sizing resolution can be obtained using electrophoresis, dynamic range is limited, requiring specific assays tailored to small (PAGE), medium (slab agarose), or large DNA (PFGE). Furthermore, detection sensitivity is limited and quantification accuracy is poor, necessitating large sample volumes and DNA amounts. Capillary electrophoresis features increased sensitivity, fast analysis times, and reduced sample consumption but with increased complexity, cost, and the same limited sizing dynamic range. Single molecule free solution hydrodynamic separation (SML-FSHS) is an integrated technique for sized-based separation of DNA and quantification by single molecule counting that performs separation with only a buffer-filled microcapillary and pressure source. This method has a unique combination of wide dynamic range (100 bp - 27 kbp), high sizing resolution and near zero sample consumption (10 pL). Furthermore, it is among the most sensitive, amplification-free detection methods available, requiring only 10-23 moles of DNA for analysis and possessing quantitative accuracy that approaches the limits imposed by molecular shot noise. In this Phase I SHIFT proposal, a microfluidic SML-FSHS platform called PicoSep will be developed that surpasses the sizing performance, detection sensitivity, and speed of the previous microcapillary systems and any current DNA sizing method. This will be accomplished through: 1) the initial demonstration of a micro/nanofluidic device to enhance speed and resolution, 2) the novel incorporation electrokinetic sample stacking to further enhance concentration sensitivity, and 3) the development of a compact instrument platform to reduce cost and facilitate ease-of-use. This instrument will address the preconceptions that single molecule methods are bulky, expensive, and difficult. The unmatched sensitivity, wide dynamic range, and low sample consumption will make PicoSep an ideal detection platform for PCR-free, multiplexed biomarker assays in clinical diagnostics and cancer medicine. PUBLIC HEALTH RELEVANCE: A Microfluidic Single Molecule Free Solution Hydrodynamic Separation Platform DNA sizing and separation is one of the most fundamental and widely used tools in molecular biology. PicoSep will have wider sizing dynamic range, higher detection sensitivity, and lower sample consumption than any existing technique. This will enable the development of new highly sensitive, multiplex biomarker assays for clinical diagnostics and cancer research that are more powerful and less expensive than current approaches.