PicoSep - A Microfluidic Single Molecule Free Solution Hydrodynamic Separation Pl

PicoSep - 微流控单分子自由溶液流体动力分离 Pl

• 批准号: 8315284
• 负责人: Kelvin Liu
• 金额: $ 19.97万
• 依托单位: CIRCULOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-15 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8315284
• 关键词: Address; Adoption; Area; Biological Assay; Biological Markers; Blood capillaries; Buffers; Cancer Diagnostics; Capillary Electrophoresis; Clinical; Computers; Consumption; Cost Control; DNA; DNA analysis; Detection; Development; Devices; Diagnostic; Electrophoresis; Equilibrium; Exclusion; Goals; Heterogeneity; High Pressure Liquid Chromatography; Medicine; Methods; Microfluidic Microchips; Microfluidics; Mole the mammal; Molecular; Molecular Biology; Noise; Performance; Phase; Publications; Pulsed-Field Gel Electrophoresis; Reproducibility; Resolution; Running; Sampling; Sepharose; Solutions; Source; Speed; System; Techniques; Time; anticancer research; base; capillary; cost; design; digital; gel electrophoresis; improved; instrument; nanofluidic; novel; pressure; prototype; single molecule; success; tool

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.

描述（由申请人提供）：微流体单分子无溶液流体动力学平台，尽管具有现代方法（例如微阵列，数字PCR和深层测序），但凝胶电泳仍然是分子生物学的工作马。虽然可以使用电泳可获得高尺寸的分辨率，但动态范围是有限的，需要针对小（页），中（平板琼脂糖）或大DNA（PFGE）量身定制的特定测定。此外，检测灵敏度有限，定量精度较差，需要大量样品体积和DNA量。毛细血管电泳具有提高灵敏度，快速分析时间和减少样本消耗，但复杂性，成本增加以及相同的有限尺寸动态范围。单分子溶液流体动力学分离（SML-FSHS）是一种集成技术，用于基于大小的DNA分离和通过单分子计数进行定量，仅使用一个充满缓冲液的微毛细管和压力来源进行分离。该方法具有广泛的动态范围（100 bp -27 kbp），高尺寸分辨率和接近零样品消耗（10 PL）的独特组合。此外，它是可用的最敏感，无扩增的检测方法之一，仅需要10-23摩尔的DNA进行分析和具有定量准确性，以接近分子射击噪声所施加的限制。在此阶段I偏移提案中，将开发一个称为PICOSEP的微流体SML-FSHS平台，该平台将超过先前微毛细管系统的尺寸性能，检测灵敏度和速度以及任何当前的DNA尺寸方法。这将通过以下方式完成：1）微/纳米富集设备的初始演示以增强速度和分辨率，2）新型掺入电动器样品堆叠以进一步增强浓度敏感性，3）3）紧凑型仪器平台的发展以降低成本，以降低成本并促进便利。该仪器将解决单分子方法笨重，昂贵且困难的先入。无与伦比的灵敏度，宽的动态范围和低样本消耗将使Picosep成为临床诊断和癌症医学中无PCR，多重生物标志物测定法的理想检测平台。 公共卫生相关性：微流体单分子的无溶液流体动力学平台DNA尺寸和分离是分子生物学中最基本和最广泛使用的工具之一。与任何现有技术相比，PICOSEP的尺寸动态范围更大，检测灵敏度较高，样品消耗较低。这将使新的高度敏感，多重生物标志物测定法进行临床诊断和癌症研究的开发，这些测定比目前的方法更强大，更便宜。