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）：微流控单分子无溶液流体动力学分离平台尽管微阵列、数字PCR和深度测序等现代方法具有强大的功能，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 DNA (PAGE)、中 DNA (平板琼脂糖) 或大 DNA (PFGE) 进行定制的特定测定。此外，检测灵敏度有限，定量精度差，需要大量样本和 DNA 量。毛细管电泳具有更高的灵敏度、更快的分析时间和更少的样品消耗，但复杂性和成本也更高，并且尺寸动态范围同样有限。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 分离和单分子计数定量的集成技术，仅使用填充缓冲液的微毛细管和压力源进行分离。该方法独特地结合了宽动态范围 (100 bp - 27 kbp)、高尺寸分辨率和接近零的样品消耗 (10 pL)。此外，它是现有的最灵敏、无扩增的检测方法之一，仅需要 10-23 摩尔 DNA 进行分析，并且具有接近分子散粒噪声限制的定量精度。在这一第一阶段 SHIFT 提案中，将开发一种名为 PicoSep 的微流控 SML-FSHS 平台，该平台的尺寸测量性能、检测灵敏度和速度将超越以前的微毛细管系统和任何当前的 DNA 尺寸测量方法。这将通过以下方式实现：1) 初步演示微/纳流体装置以提高速度和分辨率，2) 新颖的合并动电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促进易用性。该仪器将解决单分子方法体积大、昂贵且困难的先入之见。无与伦比的灵敏度、宽动态范围和低样品消耗量将使 PicoSep 成为临床诊断和癌症医学中无 PCR、多重生物标志物测定的理想检测平台。 公共健康相关性：微流控单分子无溶液流体动力分离平台 DNA 大小和分离是分子生物学中最基本和广泛使用的工具之一。与任何现有技术相比，PicoSep 具有更宽的定量动态范围、更高的检测灵敏度和更低的样品消耗。这将使开发用于临床诊断和癌症研究的新的高灵敏度、多重生物标志物检测方法成为可能，这些检测方法比现有方法更强大且更便宜。