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和深度测序的力量，凝胶电泳仍然是分子生物学的主要方法。虽然使用电泳可以获得高尺寸分辨率，但动态范围有限，需要针对小（PAGE），中（平板琼脂糖）或大DNA （PFGE）进行特定的测定。此外，检测灵敏度有限，定量准确性差，需要大样本量和DNA量。毛细管电泳具有灵敏度高、分析时间短、样品消耗少的特点，但也增加了复杂性、成本和同样有限的施胶动态范围。单分子自由溶液水动力分离（SML-FSHS）是一种基于DNA大小的分离和单分子计数定量的集成技术，它只需要一个充满缓冲液的微毛细管和压力源就可以进行分离。该方法具有宽动态范围（100 bp - 27 kbp）、高尺寸分辨率和近零样品消耗（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，并且具有接近分子射击噪声所施加的限制的定量准确性。在第一阶段的SHIFT提案中，将开发一种名为PicoSep的微流控SML-FSHS平台，该平台将超越先前的微毛细管系统和任何当前的DNA上浆方法的上浆性能，检测灵敏度和速度。这将通过以下方式实现：1)微/纳米流体装置的初步演示，以提高速度和分辨率；2)新型混合电动样品堆叠，以进一步提高浓度灵敏度；3)开发紧凑型仪器平台，以降低成本并便于使用。该仪器将解决单分子方法笨重、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、多重生物标志物检测的理想检测平台。

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.