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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
Project #
1R43GM103356-01
Application #
8315284
Study Section
Special Emphasis Panel (ZRG1-IMST-A (12))
Program Officer
Wu, Mary Ann
Project Start
2012-09-15
Project End
2014-08-31
Budget Start
2012-09-15
Budget End
2013-08-31
Support Year
1
Fiscal Year
2012
Total Cost
$199,662
Indirect Cost
Name
Circulomics, Inc.
Department
Type
DUNS #
830377581
City
Baltimore
State
MD
Country
United States
Zip Code
21211