PicoSep - A Microfluidic Single Molecule Free Solution Hydrodynamic Separation Pl

描述(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有现代方法如微阵列、数字聚合酶链式反应和深度测序的力量，凝胶电泳仍然是分子生物学的主力。虽然使用电泳法可以获得高的尺寸分辨率，但动态范围有限，需要针对小型(PAGE)、中型(平板琼脂糖凝胶)或大型DNA(PFGE)量身定做特定的分析。此外，检测灵敏度有限，定量准确度较差，需要大量的样本和DNA。毛细管电泳法具有更高的灵敏度、更快的分析时间和更少的样品消耗，但同时也增加了复杂性、成本和有限的测定动态范围。单分子自由溶液流体动力分离(SML-FSHS)是一种单分子计数法分离DNA的综合技术，只需填充缓冲液的微毛细管和压力源即可完成分离。该方法具有宽动态范围(100 BP-27 KBP)、高施胶分辨率和接近零的样品消耗(10pl)的独特组合。此外，它是现有的最灵敏的、无扩增的检测方法之一，只需要10-23摩尔的DNA进行分析，并且具有接近分子散粒噪声施加的限制的定量准确性。在这项第一阶段转移方案中，将开发一种名为PicoSep的微流控SML-FSHS平台，该平台的定尺性能、检测灵敏度和速度都超过了以前的微毛细管系统和任何当前的DNA定尺方法。这将通过以下方式实现：1)初步演示微/纳米流体设备以提高速度和分辨率，2)新加入电动样品堆积以进一步增强浓度灵敏度，以及3)开发紧凑的仪器平台，以降低成本和便于使用。这个仪器将解决单分子方法笨重、昂贵和困难的先入之见。无与伦比的灵敏度、广泛的动态范围和低样品消耗将使PicoSep成为临床诊断和癌症医学中无PCR、多路生物标记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动力分离平台DNA测定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将比任何现有技术具有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。这将为临床诊断和癌症研究开发新的高灵敏度、多重生物标记物分析，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.