We report a microfluidic platform for sizing and separation of DNA called PicoSep that is among the most sensitive to date, requiring only yoctomoles of DNA (10−24 moles) and picoliters of sample (<10 pL). The cohesive integration of cylindrical illumination confocal spectroscopy based (CICS) single molecule counting with free solution hydrodynamic separation increases detection sensitivity >103-fold over traditional laser induced fluorescence (LIF) and vastly improves quantification accuracy. Separation is performed via single molecule free solution hydrodynamic separation (SML-FSHS). SML-FSHS relies on the wall exclusion mechanism to separate DNA as it is driven down buffer filled microchannel. High separation efficiency (plate number = 105 – 106) and high sizing resolution (37 bp – 2.1 kbp) are obtained across a wide dynamic range of DNA (100 bp – 27 kbp) in a single separation. Quantitative accuracy up to the limits imposed by molecular shot noise is achieved, and a limit of detection <20 molecules is demonstrated. Through the development of PicoSep, we have significantly reduced the cost and complexity of single molecule instrumentation while achieving sizing and quantification performance that surpasses capillary electrophoresis (CE). Unlike CE, no viscous sieving matrices, high voltage power supplies, or capillary wall coatings are necessary, making devices inexpensive, simple to fabricate, and easy to incorporate into lab on a chip systems.
Single-Molecule Analysis Enables Free Solution Hydrodynamic Separation Using Yoctomole Levels of DNA