Focal volume confinement by submicrometer-sized fluidic channels

Microfluidic channels with two lateral dimensions smaller than 1 mum were fabricated in fused silica for high-sensitivity single-molecule detection and fluorescence correlation spectroscopy. The effective observation volumes created by these channels are similar to100 times smaller than observation volumes using conventional confocal optics and thus enable single-fluorophore detection at higher concentrations. Increased signal-to-noise ratios are also attained because the molecules are restricted to diffuse through the central regions of the excitation volume. Depending on the channel geometries, the effective dimensionality of diffusion is reduced, which is taken into account by simple solutions to diffusion models with boundaries. Driven by electrokinetic forces, analytes could be flowed rapidly through the observation volume, drastically increasing the rate of detection events and reducing data acquisition times. The statistical accuracy of single-molecule characterization is improved because all molecules are counted and contribute to the analysis. Velocities as high as 0.1 m/s were reached, corresponding to average molecular residence times in the observation volume as short as 10 mus. Applications of these nanofabricated devices for high-throughput, single-molecule detection in drug screening and genomic analysis are discussed.