DNA fragment sizing by single molecule detection in submicrometer-sized closed fluidic channels

The fabrication of fluidic channels with dimensions smaller than 1 μm is described and characterized in respect to their use for detection of individual DNA molecules. The sacrificial layer technique is used to fabricate these devices as it provides CMOS-compatible materials exhibiting low fluorescence background. It also allows creating microfluidics circuitry of submicrometer dimensions with great control. The small dimensions facilitate single molecule detection and minimize events of simultaneous passage of more than one molecule through the measurement volume. The behavior of DNA molecules inside these channels under an applied electrical field was first studied by fluorescence correlation spectroscopy using M13 double-stranded DNA. A linear relationship between the flow speed and applied electric field across the channel was observed. Speeds as high as 5 mm/s were reached, corresponding to only a few milliseconds of analysis time per molecule. The channels were then used to characterize a mixture of nine DNA fragments. Both the distribution and relative proportions of the individual fragments, as well as the overall concentration of the DNA sample, can be deduced from a single experiment. The amount of sample required for the analysis was ∼10 000 molecules, or 76 fg. Other potential applications of these submicrometer structures for DNA analysis are discussed.