Counting single DNA molecules in a capillary with radial focusing

For single-molecule detection, usually a small detection volume of 10 pL or less is used to improve the signal-to-noise ratio. Detection of every molecule in a sample requires that the sample be driven through a well-defined volume to facilitate laser excitation. We report a novel approach to count single DNA molecules with nearly 100% efficiency. By applying an electric field across a 40 cm long, 75 x 75 mum(2) square capillary together with hydrodynamic flow from cathode to anode, we were able to concentrate more than 95% of DNA molecules into a 10 mum region at the centre of the capillary. The YOYO-1 labelled lambda-DNA molecules were imaged with an intensified CCD camera. We found that the single DNA molecule detection efficiency in a 10(-17) M solution was 114 +/- 21%. The mobility of the DNA molecules after radial focusing was relatively constant, with relative standard deviations ranging from 0.8% to 1.4%. This allowed us to match the sampling rate to the length of the detection window to maximize counting efficiency. Analysis of a 40.2 nL injected plug of 2 x 10(-14) M lambda-DNA gave a result of 492 +/- 73 molecules, which agreed well with the estimated value of 484. This method should be generally useful for counting deformable molecules or non-spherical particles at extremely low concentrations.